Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention is related to a virus, preferably an adenovirus,
characterised in that the virus comprises: a lacking functional
wildtype E1 region, and a transporter for the transport of YB-1 into the
nucleus of a cell which is infected with the virus.

Claims:

1.-83. (canceled)

84. A virus, preferably an adenovirus, characterised in that the virus
comprises:a lacking functional wildtype E1 region, whereby the lacking
functional wildtype E1 region is protein IX-minus, anda transporter for
the transport of YB-1 into the nucleus of a cell which is infected with
the virus, andthat the virus comprises a nucleic acid coding for protein
IX and expresses protein IX.

85. The virus according to claim 84, characterised in that the lacking
functional wildtype E1A region is E1A-minus.

86. The virus according to claim 84, characterised in that the lacking
functional wildtype E1 region is E1B-minus.

87. The virus according to claim 86, characterised in that the lacking
wildtype E1 region is E1B55K-minus and/or E1B19K-minus.

88. The virus according to claim 87, characterised in that the transporter
is a transporter provided by the virus.

89. The virus according to claim 88, characterised in that the transporter
is a viral transporter.

90. The virus according to claim 84, characterised in that the transporter
comprises protein E4orf6.

91. The virus according to claim 91, characterised in that the transporter
comprises protein E1B55K.

92. The virus according to claim 88, characterised in that the transporter
comprises a complex of E4orf4 and E1B55K.

93. The virus according to claim 84, characterised in that the transporter
is coded by a nucleic acid, whereby the nucleic acid is under the control
of a promoter.

94. The virus according to claim 93, characterised in that the transporter
is a complex of at least two factors and whereby each factor is coded by
a nucleic acid, whereby both nucleic acids are controlled by a shared
promoter.

95. The virus according to claim 94, characterised in that both nucleic
acid are connected through an element which controls the expression
strength, whereby the element is preferably selected from the group
comprising IRES.

96. The virus according to claim 93, characterised in that the transporter
is a complex of at least two factors and whereby each factor is coated by
a nucleic acid, whereby both nucleic acids are controlled by a
proprietary promoter.

97. The virus according to claim 93, characterised in that the promoter is
different from the E4 promoter, in particular the adenoviral E4 promoter
and is different from the E1B promoter, in particular the adenoviral E1B
promoter.

98. The virus according to claim 93, characterised in that the promoter is
selected from the group comprising tissue-specific promoters,
tumor-specific promoters, the CMV-promoter, viral promoters and
particularly adenoviral promoters under the proviso that these are
different from the E4 promoter, the E1B promoter and preferably also
different from the E2-late promoter.

99. The virus according to claim 84, characterised in that the nucleic
acid coding for the transporter has a 3'-UTR at the 3' end of E1B55K.

100. The virus according to claim 84, characterised in that if the lacking
wildtype E1 region is EB55K-positive, the nucleic acid coding for the
transporter does not comprise an E1B55K coding nucleic acid.

101. The virus according to claim 84, characterised in that the nucleic
acid coding for the transporter codes for E1B55K and E1B19K.

102. The virus according to claim 101, characterised in that the nucleic
acid coding for the transporter codes for protein IX.

103. The virus according to claim 101, characterised in that the nucleic
acid coding for the E1B55K and E1B19K is under the control of a promoter.

104. The virus according to claim 101, characterised in that the nucleic
acid coding for the E1B55K and/or E1B19K and/or protein IX is under the
control of a promoter, whereby the promoter is different from an
E1A-dependent promoter.

105. The virus according to claim 84, characterised in that the lacking
functional wildtype E1 region is E1A13S-minus and/or E1A12S-minus.

106. The virus according to claim 84, characterised in that the lacking
functional wildtype E1 region is E1A13S-minus.

107. The virus according to claim 84, characterised in that preferably the
lacking wildtype E1 region is E1A13S-minus and E1A12-minus, whereby the
virus comprises a nucleic acid coding for the E1A12S protein, whereby the
nucleic acid is preferably a heterologous nucleic acid.

108. The virus according to claim 107, characterised in that the nucleic
acid coding for the E1A12S protein is under the control of a promoter,
whereby the promoter is preferably a YB-1 dependent promoter and more
preferably selected from the group comprising the adenoviral E2-late
promoter, the MDR-promoter and the DNA polymerase alpha promoter.

109. The virus according to claim 108, characterised in that the nucleic
acid(s) coding for the transporter code for E4orf6 and E1B55K.

110. The virus according to claim 108, characterised in that the virus
comprises a nucleic acid coding for protein IX, whereby preferably the
nucleic acid coding for E1A12S and the nucleic acid coding for protein IX
are under the control of a shared promoter, whereby more preferably both
nucleic acids are linked to each other through an expression regulating
element, whereby the element is more preferably selected from the group
comprising IRES.

111. The virus according to claim 107, characterised in that the nucleic
acid coding for the E1A12S protein and the nucleic acid coding for the
protein IX are each under the control of a promoter, whereby the promoter
is preferably the same promoter.

112. The virus according to claim 110, characterised in that the promoter
is a YB-1 dependent promoter, which is preferably selected from the group
comprising the adenoviral E2-late promoter, the MDR promoter and the DNA
polymerase-alpha promoter.

113. The virus according to claim 107, characterised in that the virus
comprises a YB-1 coding nucleic acid.

114. The virus according to claim 113, characterised in that the nucleic
acid coding for the E1A12S protein and the nucleic acid coding for the
YB-1 are under the control of a shared promoter, whereby preferably both
nucleic acids are linked to each other by an expression regulating
element, whereby the element is preferably selected from the group
comprising IRES.

115. The virus according to claim 113, characterised in that the nucleic
acid coding for YB-1 and the nucleic acid coding for E1A12S protein are
each under the control of a promoter, whereby the promoter is preferably
the same promoter.

116. The virus according to claim 113, characterised in that the promoter
is a YB-1 dependent promoter which is preferably selected from the group
comprising the adenoviral E2-late promoter, the MDR promoter and the DNA
polymerase-alpha promoter.

117. The virus according to claim 107, characterised in that the nucleic
acid coding for E1A12S is cloned into the E3 region or E4 region.

118. The virus according to claim 107, characterised in that the nucleic
acid coding for E1A12S and the nucleic acid coding for the protein IX or
the nucleic acid coding for the YB-1 are cloned into the E3 region or the
E4 region.

119. The virus according to claim 114, characterised in that the
expression of the nucleic acid coding for protein IX is controlled by a
promoter different from E1B via E1B19K or via E12AS.

120. The virus according to claim 84, characterised in that the virus
comprises at least one transgene which is preferably cloned into the E3
region.

121. The virus according to claim 120, characterised in that the virus
comprises at least one transgene which is preferably cloned into the E4
region.

122. The virus according to claim 84 comprising a nucleic acid coding for
the RGD motif, whereby the RGD motif is preferably cloned into the
HI-loop domain of the fibre knob.

124. The virus according to claim 84, characterised in that the virus is
replication deficient in cells which do not contain YB-1 in the nucleus.

125. The virus according to claim 84, characterised in that the virus can
replicate in cells which have YB-1 in the nucleus, in particular have
YB-1 in the nucleus independent of the cell cycle.

126. The virus according to claim 84, characterised in that the virus is
replication deficient in cells where or in which YB-1 is deregulated.

127. The virus according to claim 84, characterised in that the virus is
capable of replicating in tumor cells, preferably tumor cells which are
resistant against cytostatics and/or radiation.

128. The virus according to claim 127, characterised in that the cells are
multiple-drug resistant.

129. A nucleic acid coding for a virus according to claim 84 or a part
thereof.

130. Use of a virus according to claim 84 or a nucleic acid according to
claim 129 or a vector comprising the same or a replication system
comprising such nucleic acid or a part thereof, for the manufacture of a
medicament.

131. Use of a virus according to claim 84 or a nucleic acid according to
claim 129 for replication in cells, whereby the cells contain YB-1 in the
nucleus, preferably contain YB-1 in the nucleus independent of the cell
cycle, or the cells contain deregulated YB-1 or that the cells are tumor
cells, preferably tumor cells which are resistant against cytostatics
and/or radiation.

132. Use according to claim 131, characterised in that the cells contain
YB-1 in the nucleus after or due to a measure which is applied to the
cell or has been applied to the cell and is selected from the group
comprising radiation, application of cytostatics and hyperthermia.

133. Use according to claim 131, characterised in that the medicament is
for the treatment of tumors and/or cancer(s) and/or for the restoration
of sensitivity of cells to cytostatics and/or radiation, whereby
preferably the cells are tumor cells which are resistant against
cytostatics and/or radiation.

134. Use according to claim 133, characterised in that at least one part
of the cells forming the tumor are cells which have YB-1 in the nucleus,
preferably contain YB-1 in the nucleus independent of the cell cycle, or
that at least a part of the cells forming the tumor have deregulated YB-1
or at least a part of the cells forming the tumor are tumor cells, more
preferably tumor cells which are resistant against cytostatics and/or
radiation.

135. Use according to claim 134, characterised in that the cells,
particularly the cells forming the tumor or parts thereof, are resistant,
in particular multi-resistant against drugs, preferably antitumor agents
and more preferably cytostatics.

136. Use according to claim 135, characterised in that the cells show an
expression, more preferably an overexpression of the membrane bound
transport protein P-glycoprotein.

137. Use according to claim 131, characterised in that the cells have YB-1
in the nucleus, and particularly that the cells forming the tumor or part
thereof have YB-1 in the nucleus.

138. Use according to claim 131 characterised in that the tumor contains
YB-1 in the nucleus after induction of the transport of YB-1 into the
nucleus.

139. Use according to claim 139, characterised in that the transport of
YB-1 into the nucleus is triggered by at least one measure which is
selected from the group comprising radiation, application of cytostatics
and hyperthermia.

140. Use according to claim 139, characterised in that the measure is
applied to a cell, an organ or an organism.

141. Use of a virus replication system, particularly an adenoviral
replication system, comprising a nucleic acid which codes for a virus,
particularly an adenovirus, according to claim 84 or a part thereof, and
comprising a nucleic acid of a helper virus, whereby the nucleic acid of
the helper virus comprises a nucleic acid sequence which codes for YB-1,
and optionally complements the virus, preferably for the manufacture of a
medicament, more preferably for the treatment of tumors and/or cancer(s)
and/or for restoration of the sensitivity of cells to cytostatics and/or
radiation, whereby the cells are preferably tumor cells which are
resistant against cytostatics and/or radiation.

142. Use of a viral replication system according to claim 141,
characterised in that the viral nucleic acid, preferably the adenoviral
nucleic acid and/or the nucleic acid of the helper virus are present as
replicable vector.

143. Use of a nucleic acid coding for a virus, preferably an adenovirus
according to claim 84 for the manufacture of a medicament, preferably for
the manufacture of a medicament for the treatment of tumors and/or for
restoration of sensitivity of cells to cytostatics and/or radiation,
whereby the cells are preferably tumor cells which are resistant against
cytostatics and/or radiation.

144. Use according to claim 143, characterised in the cells, and
particularly the cells forming the tumor or parts thereof, are resistant,
in particular multiple-resistant against drugs, preferably antitumor
agents and more preferably cytostatics.

145. A vector comprising a nucleic acid according to claim 129.

146. Use of an agent interacting with YB-1 for the characterisation of
cells, cells of a tumor tissue or patients, in order to determine whether
such cells, cells of a tumor tissue or patients can/should be contacted
and/or treated with a virus according to claim 84 or a nucleic acid
according to claim 129.

147. Use according to claim 146, characterised in that the agent is
selected from the group comprising antibodies, high affinity binding
peptides, antikalines, aptamers, aptazymes and spiegelmers.

148. A pharmaceutical composition comprising a virus according to claim
84, or a nucleic acid according to claim 129 or a viral replication
system as described in claim 141.

149. The pharmaceutical composition according to claim 148, whereby the
composition comprises at least one further pharmaceutically active agent.

151. The pharmaceutical composition according to claim 148, characterised
in that the composition comprises a combination of at least two
compounds, whereby preferably any compound is each and independently
selected from the group comprising cytostatics.

152. The pharmaceutical composition according to claim 151, characterised
in that at least two of the compounds target different target molecules.

153. The pharmaceutical composition according to claim 152, characterised
in that at least two of the compounds are active through different modes
of action.

154. The pharmaceutical composition according to claim 151, characterised
in that at least one compound increases the infectability of a cell in
which the virus is replicating.

155. The pharmaceutical composition according to claim 151, characterised
in that at least one compound influences the availability of a compound
in the cell, preferably increases the availability of the compound,
whereby the compound mediates the uptake of the virus in one or the cell,
preferably the one in which the virus replicates.

156. The pharmaceutical composition according to claim 151, characterised
in that at least one of the compound mediates the transport of YB-1 into
the nucleus, preferably increases the same.

157. The pharmaceutical composition according to claim 151, characterised
in that at least one compound is a histone deacylase inhibitor.

158. The pharmaceutical composition according to claim 157, characterised
in that the histone deacylase inhibitor is selected from the group
comprising trichostatine A, FR 901228, MS-27-275, NVP-LAQ824, PXD101,
apicidine and scriptaid.

159. The pharmaceutical composition according to claim 151, characterised
in that at least one compound is selected from the group comprising
trichostatine A, FR 901228, MS-27-275, NVP-LAQ824, PXD101, apicidine and
scriptaid.

160. The pharmaceutical composition according to claim 151, characterised
in that at least one compound is a topoisomerase inhibitor.

161. The pharmaceutical composition according to claim 160, characterised
in that the topoisomerase inhibitor is selected from the group comprising
camptothecin, irinotecan, toptecan, DX-895If, SN-38, 9-aminocamptothecin,
9-nitrocamptothecin, daunorubicin and etoposid.

162. The pharmaceutical composition according to claim 161, characterised
in that the composition comprises trichostatine A and irinotecan.

163. The pharmaceutical composition according to claim 148, characterised
in that the virus, in particular the virus according to claim 84 is
separated from one or both or all of the at least two compounds.

164. The pharmaceutical composition according to claim 163, characterised
in that at least one unit dose of the virus is separated from at least
one unit dose of the or all further pharmaceutically active compound(s)
or from one or the at least two compounds.

165. A kit comprising a virus according to claim 163, and at least two
pharmaceutically active agents, whereby each pharmaceutically active
agent is individually and independently selected from the group
comprising cytostatics.

Description:

[0001]The invention relates to E1-minus adenoviruses and nucleic acids
coding therefore and the use thereof.

[0002]A number of therapeutic concepts are currently used in the treatment
of tumors. Apart from using surgery, chemotherapy and radiotherapy are
predominant. All these techniques are, however, associated with
considerable side effects for the patient. The use of replication
selective oncolytic viruses provides for a new platform for the treatment
of tumors. In connection therewith a selective intratumoral replication
of a virus is initiated which results in virus replication, lysis of the
infected tumor cell and spreading of the virus to adjacent tumor cells.
As the replication capabilities of the virus are limited to tumor cells,
normal tissue is spared from replication and thus from lysis by the
virus.

[0003]For the time being, several viral systems are subject to clinical
trials aiming at tumor lysis. One example for such an adenovirus is
d11520 (Onyx-015) which has been successfully used in clinical phases I
and II (Khuri, F. et al. Nature Medicine 6, 879-885, 2000). Onyx-015 is
an adenovirus having a completely deleted E1B-55kDa gene. The complete
deletion of the E1B-55kDa protein of the adenovirus is based on the
discovery that replication and thus lysis of cells is possible with an
adenoviral vector whereby the cells have a p53 deficiency (Kirn, D. et
al., Proc. Am. Soc. Clin. Oncol. 17, 391a, 1998), whereby normal cells
are not harmed. More particularly, the E1B-55kDa gene product is involved
in the inhibition of p53, the transport of viral mRNA and the switching
off of the protein synthesis of the host cell. The inhibition of p53
occurs via formation of a complex consisting of p53 and the adenoviral
encoded E1B-55kDa protein and/or a complex consisting of E1B-55kDa and
E4orf6. p53 coded by TP53 is the starting point for a complex regulatory
mechanism (Zambetti, G. P. et al., FASEB J. 7, 855-865, 1993), which
results, among others, in an efficient suppression of the cellular
replication of viruses like adenoviruses. The gene TP 53 is deleted or
mutated in about 50% of all human tumors which results in the absence of
a--desired--apoptosis due to chemotherapy or radiation therapy and thus
in an usually unsuccessful tumor treatment.

[0004]A further concept of tumorlytic adenoviruses is based on the
discovery that if the E1A protein is present in a specifically deleted
form or comprises one or several mutations, which do not affect the
binding of Rb/E2F and/or p107/E2F and/or p130/E2F, such adenovirus will
not induce the entry of the infected cells into the S phase and will be
capable of replicating in tumor cells which do not have a functional Rb
protein. Additionally, the E1A protein can be deleted at the N-terminus
and can comprise one or several mutations in the region of amino acid
positions 1 to 76 of the E1A proteins, respectively, in order to inhibit
the binding of E1A to p300 and thus to provide for a more selective
replication in tumor cells. These approaches are described in an
exemplary manner in European patent EP 0 931 830. Examples for such
viruses are AdΔ24, dl922-947, E1Ad/01/07 and CB016 (Howe, J. A. et
al., Molecular Therapy 2, 485-495, 2000; Fueyo, J. et al., Oncogene 19,
2-12, 2000; Heise, C. et al., Nature Medicine 6, 11341139, 2001; Balague,
C. et al., J. Virol. 75, 7602-7611, 2001). These adenoviral systems for
oncolysis known in the prior art thus comprise distinct deletions in the
E1A protein, whereby such deletions had been made under the assumption
that a functional Rb protein and a complex consisting of intact Rb
protein and E2F, respectively, would block an efficient in vivo
replication and in order to provide an adenoviral replication in vivo in
Rb-negative/mutated cells only. These adenoviral systems according to the
prior art are based on E1A in order to control in vivo replication by
means of the early E2 promoter (E2 early promoter) and free E2F (Dyson,
N. Genes & Development, 12, 2245-2262, 1998).

[0005]Another form of tumorlytic adenoviral systems is based on the use of
selective promoters for specifically expressing the viral oncogene E1A
which provides for a selective replication in tumor cells (Rodriguez, R.
et al., Cancer Res. 57, 2559-2563, 1997).

[0006]As described above, the selection of a cellular background which is
appropriate for the mode of action underlying the respective concept, is
important for the various concepts of adenoviral tumorlytic viruses. In
other words, the various adenoviral systems currently known may only be
used if distinct molecular biological prerequisites are realized. This
limits the use of such systems to distinct patient groups.

[0007]A particular problem in the treatment of tumor diseases arises once
the patients develop a so-called multidrug resistance (MDR) which
represents a particularly well studied form of resistance of tumors
against cytostatics (Gottesman and Pastan, Annu. Rev. Biochem. 62,
385-427, 1993). It is based on the overexpression of the membrane-bound
transport protein P-glycoprotein which belongs to the so-called ABC
transporters (Stein, U. et al., JBC 276, 28562-69, 2001, J. Wijnholds,
Novartis Found Symp., 243, 69-79, 2002). Bargou, R. C. et al. and Oda, Y.
et al (Bargou, R. C. et al., Nature Medicine 3, 447-450, 1997; Clin.
Cancer Res. 4, 2273-2277, 1998) were able to show that nuclear
localisation of the human transcription factor YB-1 is directly involved
in the activation of the expression of the P-glycoprotein. Further
studies confirmed that YB-1 is transported into the nucleus by various
stress conditions such as for example UV irradiation, administration of
cytostatics (Koike, K. et al., FEBS Lett 17, 390-394, 1997) and
hyperthermia (Stein, U. et al., JBC 276, 28562-69, 2001). Further studies
confirmed that the nuclear localisation of YB-1 has an impact on another
ABC transporter. This ABC transporter is referred to as MRP (multidrug
resistance-related protein) and is involved in the formation of the
so-called atypical, non-P-glycoprotein dependent multidrug resistance
(Stein, U. et al., JBC 276, 28562-69, 2001).

[0008]The problem underlying the present invention is to provide a
technical teaching and in particular a means which allows specifically to
treat an organism, more particularly a human organism and a group of
patients, respectively, with tumorlytically active agents. It is a
further problem underlying the present invention to provide a means which
is suitable to cause tumor lysis in patients suffering from tumor
diseases which are resistant to cytostatics, particularly those which
have a multidrug resistance. A further problem underlying the present
invention is to provide for an adenovirus which is suitable for cell
lysis. Another problem underlying the present invention was to provide a
virus which replicates in tumor cells and particularly in tumor cells
which have YB-1 in the nucleus independent of the cell cycle, or tumor
cells with deregulated YB-1, and which shows a particularly high particle
formation.

[0009]In accordance with the invention, the problem is solved by the
subject matter of the attached independent claims. Preferred embodiments
may be taken from the also attached dependent claims.

[0010]In a first aspect the problem is also solved in accordance with the
present invention by a virus, preferably an adenovirus, whereby the virus
comprises: [0011]a lacking functional wildtype E1 region, and [0012]a
transporter for the transport of YB-1 into the nucleus of a cell which is
infected with the virus.

[0013]In an embodiment of the first aspect the virus comprises a nucleic
acid coding for protein IX and expresses protein IX.

[0014]In an embodiment of the first aspect the lacking functional wildtype
E1A region is E1A-minus.

[0015]In an embodiment of the first aspect the lacking functional wildtype
E1 region is E1B-minus.

[0016]In a preferred embodiment of the first aspect the lacking wildtype
E1 region is E1B55K-minus and/or E1B19K-minus and/or protein IX-minus.

[0017]In an embodiment of the first aspect the transporter is a
transporter provided by the virus.

[0018]In a preferred embodiment of the first aspect the transporter is a
viral transporter.

[0019]In an embodiment of the first aspect the transporter comprises
protein E4orf6.

[0020]In an embodiment of the first aspect the transporter comprises
protein E1B55K.

[0021]In an embodiment of the first aspect the transporter comprises a
complex of E4orf4 and E1B55K.

[0022]In an embodiment of the first aspect the transporter is coded by a
nucleic acid, whereby the nucleic acid is under the control of a
promoter.

[0023]In a preferred embodiment of the first aspect the transporter is a
complex of at least two factors and whereby each factor is coded by a
nucleic acid, whereby both nucleic acids are controlled by a shared
promoter.

[0024]In a preferred embodiment of the first aspect both nucleic acid are
connected through an element which controls the expression strength,
whereby the element is preferably selected from the group comprising
IRES.

[0025]In a preferred alternative embodiment of the first aspect the
transporter is a complex of at least two factors and whereby each factor
is coded by a nucleic acid, whereby both nucleic acids are controlled by
a proprietary promoter.

[0026]In an embodiment of the first aspect the promoter is different from
the E4 promoter, in particular the adenoviral E4 promoter, and is
different from the E1B promoter, in particular the adenoviral E1B
promoter.

[0027]In an embodiment of the first aspect the promoter is selected from
the group comprising tissue-specific promoters, tumor-specific promoters,
the CMV-promoter, viral promoters and particularly adenoviral promoters,
under the proviso that these are different from the E4 promoter, the E1B
promoter and preferably also different from the E2-late promoter.

[0028]In an embodiment of the first aspect the nucleic acid coding for the
transporter has a 3'-UTR at the 3' end of E1B55K.

[0029]In an embodiment of the first aspect the nucleic acid coding for the
transporter does not comprise an E1B55K coding nucleic acid if the
lacking wildtype E1 region is E1B55K-positive.

[0030]In an embodiment of the first aspect the nucleic acid coding for the
transporter codes for E1B55K and E1B19K.

[0031]In an embodiment of the first aspect the nucleic acid coding for the
transporter codes for protein IX.

[0032]In a preferred embodiment of the first aspect the nucleic acid
coding for the E1B55K and E1B19K is under the control of a promoter.

[0033]In an alternative preferred embodiment of the first aspect the
nucleic acid coding for the E1B55K and/or E1B19K and/or protein IX is
under the control of a promoter, whereby the promoter is different from
an E1A-dependent promoter.

[0034]In an embodiment of the first aspect the lacking functional wildtype
E1 region is E1A13S-minus and/or E1A12S-minus.

[0035]In an embodiment of the first aspect the lacking functional wildtype
E1 region is E1A13S-minus.

[0036]In an embodiment of the first aspect preferably the lacking wildtype
E1 region is E1A13S-minus and E1A12-minus, whereby the virus comprises a
nucleic acid coding for the E1A12S protein, whereby the nucleic acid is
preferably a heterologous nucleic acid.

[0037]In a preferred embodiment of the first aspect the nucleic acid
coding for the E1A12S protein is under the control of a promoter, whereby
the promoter is preferably a YB-1 dependent promoter and more preferably
selected from the group comprising the adenoviral E2-late promoter, the
MDR-promoter and the DNA polymerase-alpha promoter.

[0038]In an embodiment of the first aspect and in particular the preceding
embodiment the nucleic acid(s) coding for the transporter code for E4orf6
and E1B55K.

[0039]In an embodiment of the first aspect and in particular the preceding
embodiment the virus comprises a nucleic acid coding for protein IX,
whereby preferably the nucleic acid coding for E1A12S and the nucleic
acid coding for protein IX are under the control of a shared promoter,
whereby more preferably both nucleic acids are linked to each other
through an expression regulating element, whereby the element is more
preferably selected from the group comprising IRES.

[0040]In an embodiment of the first aspect the nucleic acid coding for the
E1A12S protein and the nucleic acid coding for the protein IX are each
under the control of a promoter, whereby the promoter is preferably the
same promoter.

[0041]In a preferred embodiment of the first aspect the promoter is a YB-1
dependent promoter, which is preferably selected from the group
comprising the adenoviral E2-late promoter, the MDR promoter and the DNA
polymerase-alpha promoter.

[0042]In an embodiment of the first aspect the virus comprises a YB-1
coding nucleic acid.

[0043]In a preferred embodiment of the first aspect and in particular the
preceding embodiment the nucleic acid coding for the E1A12S protein and
the nucleic acid coding for the YB-1 are under the control of a shared
promoter, whereby preferably both nucleic acids are linked to each other
by an expression regulating element, whereby the element is preferably
selected from the group comprising IRES.

[0044]In a preferred alternative embodiment of the first aspect the
nucleic acid coding for YB-1 and the nucleic acid coding for E1A12S
protein are each under the control of a promoter, whereby the promoter is
preferably the same promoter.

[0045]In a preferred embodiment of the first aspect the promoter is a YB-1
dependent promoter which is preferably selected from the group comprising
the adenoviral E2-late promoter, the MDR promoter and the DNA
polymerase-alpha promoter.

[0046]In an embodiment of the first aspect the nucleic acid coding for
E1A12S is cloned into the E3 region or E4 region.

[0047]In an embodiment of the first aspect the nucleic acid coding for
E1A12S and the nucleic acid coding for the protein IX or the nucleic acid
coding for the YB-1 are cloned into the E3 region or the E4 region.

[0048]In an embodiment of the first aspect the expression of the nucleic
acid coding for protein IX is controlled by a promoter different from E1B
via E1B19K or via E12AS.

[0049]In an embodiment of the first aspect the virus comprises at least
one transgene which is preferably cloned into the E3 region.

[0050]In a preferred embodiment of the first aspect the virus comprises at
least one transgene which is preferably cloned into the E4 region.

[0051]In an embodiment of the first aspect the virus comprises a nucleic
acid coding for the RGD motif, whereby the RGD motif is preferably cloned
into the HI-loop domain of the fibre knob.

[0053]In an embodiment of the first aspect the virus is replication
deficient in cells which do not contain YB-1 in the nucleus.

[0054]In an embodiment of the first aspect the virus can replicate in
cells which have YB-1 in the nucleus, in particular have YB-1 in the
nucleus independent of the cell cycle.

[0055]In an alternative embodiment of the first aspect the virus is
replication deficient in cells where or in which YB-1 is deregulated.

[0056]In a further alternative embodiment of the first aspect the virus is
capable of replicating in tumor cells, preferably tumor cells which are
resistant against cytostatics and/or radiation.

[0057]In a preferred embodiment of the first aspect the cells are
multiple-drug or multidrug resistant.

[0058]In a second aspect the problem is solved in accordance with the
present invention by a nucleic acid coding for a virus according to the
first aspect of invention.

[0059]In a third aspect the problem is solved in accordance with the
present invention by the use of a virus according to the first aspect or
a nucleic acid according to the second aspect or a vector comprising the
same or a replication system comprising such nucleic acid or a part
thereof, for the manufacture of a medicament.

[0060]In a fourth aspect the problem is solved in accordance with the
present invention by the use of a virus according to the first aspect or
a nucleic acid according to the second aspect for replication in cells,
whereby the cells contain YB-1 in the nucleus, preferably contain YB-1 in
the nucleus independent of the cell cycle, or the cells contain
deregulated YB-1 or the cells are tumor cells, preferably tumor cells
which are resistant against cytostatics and/or radiation.

[0061]In an embodiment of the fourth aspect the cells contain YB-1 in the
nucleus after or due to a measure which is applied to the cell or has
been applied to the cell and is selected from the group comprising
radiation, application of cytostatics and hyperthermia.

[0062]In an embodiment of the third aspect the medicament is for the
treatment of tumors and/or cancer(s) and/or for the restoration of
sensitivity of cells to cytostatics and/or radiation, whereby preferably
the cells are tumor cells which are resistant against cytostatics and/or
radiation.

[0063]In an embodiment of the third aspect at least one part of the cells
forming the tumor are cells which have YB-1 in the nucleus, preferably
contain YB-1 in the nucleus independent of the cell cycle, or at least
one part of the cells forming the tumor have deregulated YB-1 or at least
one part of the cells forming the tumor are tumor cells, more preferably
tumor cells which are resistant against cytostatics and/or radiation.

[0064]In a preferred embodiment of the third aspect the cells,
particularly the cells forming the tumor or parts thereof, are resistant,
in particular multi-resistant against drugs, preferably antitumor agents
and more preferably cytostatics.

[0065]In an embodiment of the third and fourth aspect the cells show an
expression, more preferably an overexpression of the membrane bound
transport protein P-glycoprotein.

[0066]In an embodiment of the third and fourth aspect the cells have YB-1
in the nucleus, and particularly the cells forming the tumor or part
thereof have YB-1 in the nucleus.

[0067]In an embodiment of the third and fourth aspect the tumor contains
YB-1 in the nucleus after induction of the transport of YB-1 into the
nucleus.

[0068]In a preferred embodiment of the third and fourth aspect the
transport of YB-1 into the nucleus is triggered by at least one measure
which is selected from the group comprising radiation, application of
cytostatics and hyperthermia.

[0069]In a preferred embodiment of the third and fourth aspect the measure
is applied to a cell, an organ or an organism.

[0070]In a fifth aspect the problem is solved according to the invention
by the use of a virus replication system, particularly an adenoviral
replication system, comprising a nucleic acid which codes for a virus,
particularly an adenovirus, according to the first aspect or a part
thereof, and comprising a nucleic acid of a helper virus, whereby the
nucleic acid of the helper virus comprises a nucleic acid sequence which
codes for YB-1, and optionally complements the virus, preferably for the
manufacture of a medicament, more preferably for the treatment of tumors
and/or cancer(s) and/or for restoration of the sensitivity of cells to
cytostatics and/or radiation, whereby the cells are preferably tumor
cells which are resistant against cytostatics and/or radiation.

[0071]In an embodiment of the fifth aspect the viral nucleic acid,
preferably the adenoviral nucleic acid and/or the nucleic acid of the
helper virus are present as a replicable vector.

[0072]In a sixth aspect the problem is solved according to the invention
by the use of a nucleic acid coding for a virus, preferably an adenovirus
according to the first aspect for the manufacture of a medicament,
preferably for the manufacture of a medicament for the treatment of
tumors and/or for restoration of sensitivity of cells to cytostatics
and/or radiation, whereby the cells are preferably tumor cells which are
resistant against cytostatics and/or radiation.

[0073]In an embodiment of the sixth aspect the cells, and particularly the
cells forming the tumor or parts thereof, are resistant, in particular
multiple-resistant against drugs, preferably antitumor agents and more
preferably cytostatics.

[0074]In a seventh aspect the problem is solved according to the invention
by a vector comprising a nucleic acid according to the second aspect,
preferably for the use according to the third and fourth aspect.

[0075]In eighth aspect the problem is solved according to the invention by
the use of an agent interacting with YB-1 for the characterisation of
cells, cells of a tumor tissue or patients, in order to determine whether
such cells, cells of a tumor tissue or patients can/should be contacted
and/or treated with a virus, in particular an adenovirus, according to
the first aspect or a nucleic acid according to the second aspect.

[0076]In an embodiment of the eighth aspect the agent is selected from the
group comprising antibodies, high affinity binding peptides, antikalines,
aptamers, aptazymes and spiegelmers.

[0077]In a ninth aspect the problem is solved according to the invention
by a pharmaceutical composition comprising a virus according to the first
aspect, or a nucleic acid according to the second aspect or a viral
replication system as described in the fifth aspect.

[0078]In an embodiment of the ninth aspect the composition comprises at
least one further pharmaceutically active agent.

[0080]In an embodiment of the ninth aspect the composition comprises a
combination of at least two compounds, whereby preferably any compound is
each and independently selected from the group comprising cytostatics.

[0081]In a preferred embodiment of the ninth aspect at least two of the
compounds target different target molecules.

[0082]In an embodiment of the ninth aspect at least two of the compounds
are active through different modes of action.

[0083]In an embodiment of the ninth aspect at least one compound increases
the infectability of a cell in which the virus is replicating.

[0084]In an embodiment of the ninth aspect at least one compound
influences the availability of a compound in the cell, preferably
increases the availability of the compound, whereby the compound mediates
the uptake of the virus in one or the cell, preferably the one in which
the virus replicates.

[0085]In an embodiment of the ninth aspect at least one of the compound
mediates the transport of YB-1 into the nucleus, preferably increases the
same.

[0086]In an embodiment of the ninth aspect at least one compound is a
histone deacylase inhibitor.

[0087]In a preferred embodiment of the ninth aspect the histon deacylase
inhibitor is selected from the group comprising trichostatine A, FR
901228, MS-27-275, NVP-LAQ824, PXD101 apicidine and scriptaid.

[0088]In an embodiment of the ninth aspect at least one compound is
selected from the group comprising trichostatine A, FR 901228, MS-27-275,
NVP-LAQ824, PXD101 apicidine and scriptaid.

[0089]In an embodiment of the ninth aspect at least one compound is a
topoisomerase inhibitor.

[0090]In a preferred embodiment of the ninth aspect the topoisomerase
inhibitor is selected from the group comprising camptothecin, irinotecan,
toptecan, DX-895If, SN-38, 9-aminocamptothecin, 9-nitrocamptothecin,
daunorubicin and etoposide.

[0091]In an embodiment of the ninth aspect the composition comprises
trichostatine A and irinotecan.

[0092]In an embodiment of the ninth aspect the virus, in particular the
virus according to the first aspect of the present invention is separated
from one or both or all of the at least two compounds.

[0093]In a preferred embodiment of the ninth aspect at least one unit dose
of the virus is separated from at least one unit dose of the or all
further pharmaceutically active compound(s) or from one or the at least
two compounds.

[0094]In a tenth aspect the problem is solved according to the invention
by a kit comprising a virus, preferably a virus according to the first
aspect of the present invention, and at least two pharmaceutically active
agents, whereby each pharmaceutically active agent is individually and
independently selected from the group comprising cytostatics.

[0095]The present invention is based on the surprising finding that the
viruses according to the invention, i.e. viruses which lack a functional
E1 region as present in the wildtype adenovirus, and which, at the same
time, comprise a transporter and in particular code for a transporter
which is capable of transporting or translocating YB-1 into the nucleus,
is capable of replicating in cells which contain YB-1 in the nucleus
independent of the cell cycle, or in cells which have deregulated YB-1.

[0096]Additionally the present inventor has found that the viruses
according to the invention may also replicate independently of E1A13S, in
particular if the replication is mediated by YB-1. In connection
therewith, the replication occurs in particular in those cells as
described above. As used herein, cells which contain YB-1 in the nucleus,
preferably contain YB-1 in the nucleus independent of the cell cycle,
also comprise those which contain YB-1 in the nucleus due to the use of
the viruses according to the invention and in particular due to the
infection of the cells with them.

[0097]Finally, the present inventor has found that protein IX is an
important factor, in particular for the effectiveness of the viruses
according to the invention when used as oncolytic viruses and that this
factor is expressed by the constructs disclosed herein which results in a
high particle formation also in YB-1 mediated E1A13S independent viral
replication.

[0098]Cells which contain YB-1 in a deregulated form are those which have
at least one of the following characteristics and/or those which contain
YB-1, whereby YB-1 exhibits at least one of the following
characteristics: (1) YB-1 is overexpressed in the cells, preferably
overexpressed independent of the cell cycle, whereby preferably as a
measure for the expression it is referred to the expression of YB-1 in
normal cells, i.e. cells which are different from tumor cells or cells
and cell lines, respectively such as the following ones: hepatocytes as
well as fibroblast cell lines WI38 and CCD32-Lu. Preferably an
overexpression is an expression which is increased by the factor of about
2 to 10, preferably 5 to 10. Methods for measuring the expression and in
particular the overexpression are known to the persons skilled in the art
and comprise, among others, the measuring the protein concentration, in
particular of YB-1, measuring the RNA, in particular RNA of YB-1, Western
Blot Analysis, Northern Blot Analysis and RT-PCR, each preferably related
to or of YB-1. Rather than YB-1 also surrogate markers as described
herein may be used. Examples for cell lines which show an overexpression
of YB-1 are the following ones: colon carcinoma cell line 257RDB,
pancreas carcinoma cell line 181RDB, mamma carcinoma cell line MCF-7Adr,
prostate carcinoma cell line DU145, prostate carcinoma cell line PC3,
glioma cell line U373, glioma cell line U87, lung carcinoma cell line
A549, liver carcinoma cell lines Hep3B and HepG2. YB-1 which is present
in the cell, allows the replication of the viruses according to the
invention. It is preferred within the present invention that the
replication efficacy under such conditions is different from a strongly
reduced replication.

[0099]As used herein in an embodiment the term functional wildtype E1
region refers in particular to an E1 region as contained in the wildtype
adenovirus Ad5. In an embodiment the term lacking functional wildtype E1
region refers to an E1 region which does not contain or not completely
contain one or several of the functions and functionalities contained in
adenoviruses of the wildtype. Functionality or function, generally
referred to herein in the following as function, is represented or
mediated by a nucleic acid or a protein, preferably a protein.

[0100]In connection with the present invention the lack of the function
may be caused by the function not being active at the translation level,
i.e. that the protein mediating the function is not present, although the
nucleic acid coding therefore is still present in the viral genome. This
may, for example, be achieved by the translation controlling regulatory
elements not being active, preferably not being in the regulatory and
controlling context as present in viruses of the wildtype for the
respective feature, whereby such regulatory elements can, for example, be
present at the 3'UTR of the mRNA, which, among others, provides for the
stability of the mRNA.

[0101]In connection with the present invention the lack of the function
can alternatively or additionally be caused by the function not being
active at the transcription level, i.e. the function mediating protein is
not present and that the nucleic acid coding therefore is not contained
or not completely contained in the viral genome. It is within this
embodiment that the nucleic acid contains one or several mutations which
result in the loss of function. Such mutations are preferably point
mutations and/or deletions comprising several bases and/or a complete
deletion of the open reading frame or of the nucleic acid coding for the
protein.

[0102]A function is lacking in the sense of the above if the protein does
not exhibit all of the functions or activities of the corresponding
protein of the wildtype. In an embodiment the measure for the activity is
the extent of replication which is achieved under such conditions,
whereby it is preferably different from a strongly different replication.

[0103]In a preferred embodiment of the present invention the function is
also lacking when the function is, compared to the wildtype virus,
contained in the virus in a different regulatory context. A different
regulatory context is in an embodiment one, whereby the function is
expressed, compared to other functions, at a different point in time
and/or is under the control of a different element which controls or
influences transcription and/or translation. Such an element is in a
particular embodiment the promoter.

[0104]The lack of a function in the above sense is also indicated herein
by referring to the respective function as "minus". For example, the lack
of E1A13S is referred to as E1A13S-minus.

[0105]In an embodiment a strongly reduced replication is in particular a
replication which is reduced, compared to the wildtype, by a factor of 2,
preferably by a factor of 5, more preferably by a factor of 10 and most
preferably by a factor of 100. In a preferred embodiment the comparison
of the replication is performed using identical or similar cell lines,
identical or similar virus titers for the infection (multiplicity of
infection, MOI, or plaque forming unit, pfu) and/or identical or similar
assay conditions. Replication means in particular particle formation. In
further embodiments the measure for replication can be the extent of
viral nucleic acid synthesis. Methods for determining the extent of the
synthesis of viral nucleic acids are known to the persons skilled in the
art as are methods for the determination of particle formation.

[0106]The viruses according to the present invention comprise a
transporter for the transport of YB-1 into the nucleus. In a preferred
embodiment the transporter is a protein, preferably a viral protein. YB-1
which is transported by the transporter into the nucleus of the cell, is
one which is preferably a deregulated YB-1, in particular as defined
herein. However, it is also within the present invention that YB-1 is a
YB-1 which, alternatively or in addition to deregulated YB-1, is encoded
by the virus of the invention and is expressed by said virus in the cell
which is infected by said virus.

[0107]The cells in which the viruses of the invention transport YB-1 into
the nucleus, are preferably those which contain deregulated YB-1.

[0108]It is within the skills of the persons of the art to determine
whether a virus comprises such a transporter or is coding therefore. In
an embodiment a cell may be used whereby such cell does not contain YB-1
in a cell cycle independent manner in the nucleus such as the cervix
carcinoma cell line HeLa or the osteosarcoma cell line U2OS, and it can
subsequently be determined whether due to the infection and the
subsequent replication of the virus the thus infected cell contains YB-1
in the nucleus. In an alternative embodiment the cell used is a cell
which contains deregulated YB-1. The detection of YB-1 in the nucleus
under such experimental conditions may be performed by a person skilled
in the art by using the means described herein, in particular by using an
antibody directed against YB-1. If, under the influence of the virus,
YB-1 is detected in the nucleus, the tested virus comprises a or the
transporter.

[0109]It is within the present invention that the E1 region, with regard
to one or both protein groups which are encoded in the E1 region, is
"minus" in the sense of the above. The two protein groups are the group
of the E1A proteins, in particular the E1A 13S protein, also referred to
herein as E1A 13S, and the E1A12S protein, also referred to herein as
E1A12S, and the group of the E1B proteins, in particular the E1B55K
protein, also referred to herein as E1B55K, the E1B19K protein, also
referred to herein as E1B19K, and protein IX.

[0110]It is within an embodiment of the present invention that the virus
is E1A13S minus, if it is under the control of a promoter which is
different from the E1A promoter, preferably the adenoviral E1A promoter
and more preferably the adenoviral E1A promoter of the wildtype; that the
virus is E1A12S-minus if it is under the control of a promoter which is
different from the E1A promoter, preferably the adenoviral E1A promoter
and more preferably the adenoviral E1A promoter of the wildtype; that the
virus is E1B55K-minus, if it is under the control of a promoter which is
different from the E1B promoter, preferably the adenoviral E1A promoter
and more preferably the adenoviral E1B promoter of the wildtype; that the
virus is E1B19K-minus, if it is under the control of a promoter which is
different from the E1B promoter, preferably the adenoviral E1B promoter
and more preferably the adenoviral E1B promoter of the wildtype; and that
it is protein IX-minus if it is under the control of a promoter which is
different from the E1BIX promoter, preferably the adenoviral E1BIX
promoter and more preferably the adenoviral E1BIX promoter of the
wildtype and, while it is under the control of the E1BIX promoter, the
promoter is inactive due to lack of viral factors in particular, which
control the activity of the E1BIX promoter; the latter is thus an example
that the regulatory context has been changed, more specifically that the
regulatory context is indirectly changed or changed at a higher
integration or regulatory level. In general, the term changed regulatory
context also comprises changes which are active indirectly or at a higher
integration or regulatory level, however, in any case are different from
the circumstances of the wildtype, in particular of the wildtype
adenovirus.

[0111]In an embodiment of the present invention the virus is E1A13S-minus.
In a further embodiment the virus is also E1A12-minus. It is particularly
preferred if the viral E1A12S is under the control of a promoter the
activity of which is controlled by YB-1, in particular is activated by
YB-1. These promoters are referred to herein also as YB-1-dependent
promoters. A particularly preferred YB-1 dependent promoter is the
adenoviral E2-late promoter. By this construction it is ensured that
E1A12S is activated in viral replication only once YB-1 is contained in
the nucleus. This is achieved in case of cells having deregulated YB-1 by
the transporter of the virus according to the present invention which
translocates the deregulated YB-1 into the nucleus of the infected cell.
Due to the, compared to the expression in the wildtype, chronologically
reversed expression of the viral transporters and of E1A12S the
specificity of the expression of E1A12S is ensured in only those cells
which contain YB-1 in deregulated form and thus the replication of the
virus and, consequently, lysis is limited to these very cells which is,
from a safety point of view, an essential advantage of this construction
of the viruses.

[0112]Having this in mind the particle number was to be increased in the
YB-1 dependent replication. The present inventor has recognised that also
in YB-1 dependent replication protein IX plays an important role and that
its expression remains unchanged by the aforedescribed chronological
change of the expression of the transporter which is preferably provided
by the proteins of the E1B region, and that E1A12S is not affected if the
constructs disclosed herein are realised. The adenoviral constructs
described in the prior art for the YB-1 dependent replication showed
despite outstanding oncolytical characteristics a particle formation
which is low for some applications which, for example, requires another
application of the oncolytic virus. Such another application of viruses
is, in principle, possible, however, not desired in most cases. With the
constructs described herein, particle formation could be significantly
increased. Insofar the present invention is related to the use of protein
IX and/or a nucleic acid coding therefore, for the formation of viral, in
particular adenoviral particles in connection with YB-1 dependent
replicating viruses, in particular adenoviruses. Furthermore, the present
invention is related to the use of viruses, preferably adenoviruses,
which are replicating in a YB-1 dependent manner for the manufacture of a
medicament, whereby the viruses comprise protein IX and/or a nucleic acid
coding therefor. In a particularly preferred embodiment the medicament is
for the treatment of tumors and tumor diseases as described herein. In a
further, additional or alternative embodiment the medicament is for
reversing a resistance in animal cells as described herein and/or for the
restoration of the sensitivity of the cells towards cytostatics and/or
radiation, whereby the cells are preferably tumor cells which show a
resistance, particularly one as described herein, preferably a resistance
against cytostatics and radiation, as in particular described herein.

[0113]It is within the present invention that the various transgenes are
cloned at appropriate sites within the viral genome. Particularly
preferred are the regions E1, E2A, E2B, E3 and E4. The cloning of the
transporters into the E1 region is particularly preferred. It will be
appreciated by the persons skilled in the art that the cloning of these
transgenes into said sites of the viral genome can partially or
completely inactivate or delete the genes coded by such sites. It is,
however, also within the present invention that the genes coded by the
respective site may partially or completely remain active.

[0114]The viruses of the invention are preferable adenoviruses.

[0115]The term treatment of a disease or condition comprises in a
preferred embodiment also prevention of such disease or condition.

[0117]The virus Xvir03-3'UTR, which is as such described in the prior art
and is replicating in a YB-1 dependent manner, contains, as shown by
analysis performed in connection with the present invention, both the
promoter as well as the sequence for protein IX, as the 3'-UTR sequence
contains the same. In tumor cells, however, the protein is only weakly
expressed and results in a particle formation which is comparatively
lower than the one of wildtype virus. The virus Xvir 03-03'UTR expresses
the viral proteins E1B55k and E4orf6 by means of the heterologous CMV
promoter (Clontech: Plasmid pShuttle) which is introduced into Xvir
03-03'UTR. Rather than the CMV promoter also those promoters as disclosed
herein in connection with the expression of E1A may be used. The open
reading frames of both genes are linked to each other by a so-called IRES
sequence (internal ribosomal entry site) (Pelletier, J. and Sonenberg, N.
Nature, 1988, 334, 320-325). This element (Novagen: pCITE) allows the
expression of two proteins from one mRNA. A further option of the
expression of two proteins from one RNA is the use of short peptides (2A)
which are derived from food and mouth disease virus (Pablo de Felipe,
Genetic Vaccines and Therapy, 2004, 2, 13). This element can, basically,
be used in the various embodiments described herein as an alternative to
the regulatory IRES sequence.

[0118]With regard to this regulatory background of the expression of
protein IX in connection with YB-1 dependent replicating viruses and in
particular adenoviruses which has been unknown prior to the filing of the
present invention, the present inventor has realised that the expression
of protein IX may, in principle, be ensured in YB-1 dependent replication
and by viruses which replicate in a YB-1 dependent manner, by the
following strategies:

1. By an independent promoter, particularly one by which protein E1A12S or
protein E1B19K is controlled.

[0119]The independent promoter is preferably one which is different from
the E1BIX promoter. Preferably, the independent promoter is selected from
the group comprising tissue-specific, tumor-specific, YB-1-specific and
viral promoters.

2. Control of the expression of protein IX by E1A12S. By the expression of
the E1A12S protein an S phase induction of the infected cell occurs which
results in the activation of protein IX by its natural promoter.

[0120]It is within the present invention that, in principle, promoters are
used for the expression of the transporter which are different from the
promoter which controls the expression of the transporter in the wildtype
virus. In preferred embodiments this means that E1B55K is controlled by a
promoter different from E1B, E4orf6 by a promoter different from the E4
promoter. In a further embodiment the promoter is one which is E1A
independent, i.e. the activity of which is not influenced by E1A.
Preferred promoters are thus tissue-specific promoters, tumor-specific
promoters and viral promoters and in particular non-adenoviral vectors,
preferably those described herein.

[0121]YB-1 dependent promoters which may be used in the present invention,
include, but are not limited to: the adenoviral E2-late promoter, the MDR
promoter [Stein et al, J. Biol. Chem, 2001, 276, 28562-28569;] as well as
the DNA polymerase-alpha promoter [En-Nia et al, J. Biol. Chem., 2004,
Epub ahead of print].

[0123]From the telomerase promoter it is known that it is essential to
human cells. The telomerase activity is controlled by the transcription
control of the telomerase reverse transcriptase gene (hTERT) which is the
catalytic subunit of the enzyme. The expression of the telomerase is
active in 85% of human tumor cells. In contrast thereto it is inactive in
most normal cells with the exception of germ cells and embryonal tissue
(Braunstein, I. et al., Cancer Research, 61, 5529-5536, 2001; Majumdar,
A. S. et al., Gene Therapy 8, 568-578, 2001). More detailed analyses of
the hTERT promoter have shown that fragments of the promoter being 283 bp
and 82 bp, respectively, away from the starting codon are sufficient for
a specific expression in tumor cells (Braunstein I. et al.; Majumdar A S
et al., supra). Therefore, this promoter and the specific fragments,
respectively, are suitable for providing specific expression of a gene
and in particular a transgene, preferably a transgene as disclosed
herein, in tumor cells only.

[0124]Such a promoter is also to allow the expression of the modified
oncogene, preferably the E1A oncogene protein, of the virus of the
invention in tumor cells only. Also, in an embodiment the transgene, in
particular one which is selected from the group comprising E4orf6,
E1B55kD, ADP and YB-1 is expressed in an adenoviral vector under the
control of one of these promoters. It is within the present invention
that the reading frame of the transactivating oncogene protein, in
particular of the E1A protein is in frame with one or several of the gene
products of the adenoviral system. The reading frame of the
transactivating E1A protein may, however, also be independent therefrom.

[0125]As used herein, the term transgene refers, in an embodiment, to all
those genes which are either not contained in the virus, particularly the
adenovirus of the wildtype and more preferably the adenovirus Ad5
wildtype, or in a different regulatory context, particularly as defined
herein. The gene which is present in such a different regulatory context
is herein also referred to as heterologous gene. It is within an
embodiment of the present invention that one or several transgenes, as
described herein, are coded and/or expressed by one or more of the helper
genes.

[0126]The insights, methods, uses or nucleic acids, proteins, replication
systems and the like, respectively, described herein are not necessarily
limited to adenoviruses. In principle such systems exist also in other
viruses which are encompassed herewith.

[0127]The use of the viruses according to the invention or the use in
accordance with the present invention of the viruses described herein,
may result in a replication comparable to the one of wildtype at an
infection rate of 1 to 10 pfu/cell, compared to 10 to 100 pfu/cell
according to the prior art.

[0128]The viruses according to the present invention allow a significantly
increased particle formation compared to the YB-1 dependent viruses of
the prior art. Preferably, particle formation is increased by the factor
of 2 to 50, preferably the factor 10 to 50.

[0129]Finally, in an embodiment the adenoviruses used in accordance with
the present invention are E1B deficient, in particular E1B 19K deficient.
Deficient as generally used herein means a condition where E1B does not
show the entirety of characteristics inherent to the wildtype and at
least one of these characteristics is missing. The adenoviral BCL2
homologue E1B19k prevents E1A induced apoptosis by interaction with the
pro-apoptotic proteins Bak and Bax. Because of this a maximum replication
and/or particle formation is possible in infected cells (Ramya
Sundararajan and Eileen White, Journal of Virology 2001, 75, 7506-7516).
The lack of E1B 19k results in a better release of the viruses as, if
present, it minimises the function of the adenoviral death protein. By
such a deletion the cytopathic effect induced by the virus is increased
(Ta-Chiang Liu et al., Molecular Therapy, 2004) and thus results in an
enhanced lysis of the infected tumor cells. Additionally, the lack of
E1B19K results in TNF-alpha not having an impact on the replication of
such recombinant adenoviruses in tumor cells, whereas in normal cells the
treatment with TNF-alpha results in decreased replication and release of
infectious viruses. Thus selectivity and specificity is increased
(Ta-Chiang Liu et al., Molecular Therapy 2004, 9, 786-803).

[0130]It is within the skills of the persons of the art to delete and
mutate, respectively, adenoviral nucleic acid sequences which are
non-essential for the invention. Such deletions may, for example, be
related to nucleic acid coding for a part of the E3 and E4 as also
described herein. In case E4 is deleted it is particularly preferred if
such deletion does not extend to the protein E4orf6, in other words the
adenovirus to be used in accordance with the present invention codes
E4orf6. In preferred embodiments such adenoviral nucleic acids may still
be packed into the viral capsid and thus infectious particle be formed.
This applies equally to the use of the nucleic acids in accordance with
the present invention. Generally it has also still to be noted that the
adenoviral systems may be deficient with regard to single or several
expression products. In connection therewith, it is to be noted that this
may be based on the complete deletion or mutation of the nucleic acid
coding for the expression product or on a deletion or mutation of such
nucleic acid such that essentially no expression product is formed any
longer or on the lack of regulatory and expression-controlling,
respectively, elements such as promoters or transcription factors or on
an activity of the same which is different from wildtype, be it at the
level of the nucleic acids (lack of a promoter; cis acting element) or at
the level of the translation and transcription system, respectively
(trans-acting elements). Particularly the latter aspect may strongly
depend on the respectively cellular background.

[0131]The YB-1 dependent replication of the viruses according to the
invention occurs with regard to the replication of the adenoviruses of
the wildtype as described in the following.

[0132]The replication of adenoviruses is a very complex process and is
usually based on the human transcription factor E2F. During viral
infection at first the "early genes" E1, E2, E3 and E4 are expressed. The
group of the "late genes" is responsible for the synthesis of the
structural proteins of the virus. The E1 region consisting of two
transcriptional units E1A and E1B which code for different E1A and E1B
proteins, plays a critical role for the activation of both the early and
the late genes, as they induce the transcription of the E2, E3 and E4
genes (Nevins, J. R., Cell 26, 213-220, 1981). Additionally, the E1A
proteins may initiate DNA synthesis in resting cells and thus trigger
their entry into the S phase (c. f. Boulanger and Blair, 1991).
Additionally, they interact with the tumor suppressors of the Rb class
(Whyte, P. et al., Nature 334, 124-127, 1988). In doing so, the cellular
transcription factor E2F is released. The E2F factors may subsequently
bind to corresponding promoter regions of both cellular and viral genes
(in particular to the adenoviral E2 early promoter) and initiate
transcription and thus replication (Nevins, J. R., Science 258, 424-429,
1992).

[0133]The gene products of the E2 region are especially needed for the
initiation and completion of the replication as they code for three
essential proteins. The transcription of the E2 proteins is controlled by
two promoters, the "E2 early E2F dependent" promoter, which is also
referred to herein as E2-early promoter or early E2 promoter, and the
"E2-late" promoter (Swaminathan and Thimmapaya, The Molecular Repertoire
of Adenoviruses III: Current Topics in Microbiology and Immunology, vol.
199, 177-194, Springer Verlag 1995). Additionally, the products of the E4
region together with the E1A and E1B-55kDa protein play a crucial role
for the activity of E2F and the stability of p53. For example, the E2
promoter is even more transactivated by direct interaction of the
E4orf6/7 protein encoded by the E4 region, with the heterodimer
consisting of E2F and DP1 (Swaminathan and Thimmapaya, J B C 258,
736-746, 1996). Furthermore, the complex consisting of E1B-55kDa and
E4orf6 is inactivated by p53 (Steegenga, W. T. et al., Oncogene 16,
349-357, 1998) in order to complete a successful lytic infectious cycle.
Additionally, E1B-55kDa has a further important function insofar as it
promotes, when interacting with E4orf6 protein, the export of viral RNA
from the nucleus, whereas cellular RNAs are retained in the nucleus
(Bridge and Ketner, Virology 174, 345-353, 1990). A further important
observation is that the protein complex consisting of E1B-55kDa/E4orf6 is
localised in the so-called "viral inclusion bodies". It is assumed that
these structures are the sites of replication and transcription (Ornelles
and Shenk, J. Virology 65, 424-429, 1991).

[0134]The E3 region is another important region for the replication and in
particular for the release of adenoviruses. The E3 region more precisely
contains the genetic information for a variety of comparatively small
proteins which are not essential for the infectious cycle of adenovirus
in vitro, i.e. in cell culture. However, they play a crucial role in the
survival of the virus during an acute and/or latent infection in vivo as
they have, among others, immunoregulatory and apoptotic function(s)
(Marshall S. Horwitz, Virologie, 279, 1-8, 2001; Russell, supra). It
could be shown that a protein having a size of about 11.6 kDa induces
cell death. This protein was, due to its function, named ADP--for the
english term adenovirus death protein--(Tollefson, J. Virology, 70,
2296-2306, 1996). The protein is predominantly formed in the late phase
of the infectious cycle. Furthermore, the overexpression of the protein
results in a better lysis of the infected cells (Doronin et al., J.
Virology, 74, 6147-6155, 2000). In accordance therewith, the genes and
proteins, respectively, are still contained in the virus in accordance
with the present invention.

[0135]The use of the adenoviruses according to the present invention as
medicaments and in particular in connection with systemic administration
can be improved by a suitable targeting of the adenoviruses. The
infection of tumor cells by adenovirus depends, among others, to a
certain extent on the presence of the coxsackievirus-adenovirus receptor
CAR and particular integrins. If these are strongly expressed in cells,
in particular tumor cells, an infection is already possible at very low
titers (pfu/cell). Different strategies have so far been followed in
order to achieve a so called re-targeting of the recombinant adenoviruses
by, for example, insertion of heterologous sequences into the fiber knob
region and the C-terminus of protein IX, use of bi-specific antibodies,
coating of the adenoviruses with polymers, introduction of ligands in the
Ad fibre, substitution of the serotype 5 knob and serotype 5 fiber shaft
and knob, respectively, by the serotype 3 knob and Ad35 fiber shaft and
knob and modification of the penton base (Nicklin S. A. et al., Molecular
Therapy 2001, 4, 534-542; Magnusson, M. K. et. al., J. of Virology 2001,
75, 7280-7289; Barnett B. G. et al., Biochimica et Biophysica Acta 2002,
1575, 1-14; Dimitrev I P et al., Journal of Virology, 2002, 76,
6893-6899; Mizuguchi und Hayakawa, Human Gene Therapy, 2004, 15,
1034-1044). Realizing such further designs and characteristics,
respectively, in connection with the adenoviruses according to the
present invention and the adenoviruses to be used in accordance with the
invention, in their various aspects of the present invention, is within
the present invention.

[0136]The various transgenes, including E1B55kD, E4orf6, ADP and the like,
in particular if they are viral genes, may in principle be cloned from
any respective virus, preferably adenovirus and more preferably
adenovirus Ad5. A variety of plasmids are additionally described in the
prior art which contain the respective genes and from which these may
accordingly be taken and introduced into both the adenoviruses according
to the present invention as well as the viruses to be used in accordance
with the present invention. An example for a plasmid expressing E1B55kD
is, for example, described by Dobbelstein, M. et al., EMBO Journal, 16,
4276-4284, 1997. The coding region of the E1B55K gene can, for example,
can be excised together with the 3' non-coding region (this 3'UTR region
lies preferably at about base position 3507-4107 of the adenovirus
wildtype genome) of this gene by means of Bam HI from the plasmid
pDCRE1B. The respective fragment comprising the E1B55kD gene as well as
the 3' non-coding region corresponds to nucleotides 2019 to 4107 of the
adenovirus type 5. It is, however, also within the present invention that
the E1B55kD gene is excised from the plasmid by means of the restriction
enzymes Bam HI and BfrI and XbaI, respectively, and subsequently cloned
into the adenovirus. It is also within the present invention that also
analogues thereof and in particular analogues of the 3' UTR region may be
used within the present invention. An analogue of the 3' UTR region is
any sequence which has the same effect as the 3' UTR region, particularly
the same effect with regard to the expression of a gene, preferably the
E1B55kD gene. Such analogues can be determined by routine experiments
performed by the persons skilled in the art, e.g. by extending or
shortening the 3' UTR region by one or several nucleotides and
subsequently testing whether the thus obtained analogue still has the
same effect as the 3' UTR region as described previously. In an
embodiment the term 3' UTR region thus comprises also each and any
analogue thereof.

[0137]Those viruses where therapeutic genes or transgenes are cloned
preferably under the control of a specific promoter, in particular a
tumor-specific or tissue-specific promoter, are further developments of
the viruses according to the present invention. It is also within such
viruses that also the E4 region is functionally inactive and is
preferably deleted. The transgenes described herein can also be cloned
into the E4 region, whereby this may be performed alternatively or
additionally to the cloning of the transgenes into the E3 region and the
E3 region may remain partially or completely intact, respectively.
Transgenes as used herein may be therapeutic genes or viral genes,
preferably adenoviral genes, which are preferably not present in the
genome of wildtype adenoviruses and which are not present, respectively,
at the site of the genome at which they are located in the particular
virus now.

[0142]In an embodiment the anti-apoptosis factors are selected from the
group comprising BCL2 and comprise also the nucleic acids coding
therefor. In an embodiment the oncogenes are selected from the group
comprising Ras, particularly mutated Ras, Rb and Myc, and comprises also
the nucleic acids coding therefor. In an embodiment the angiogenesis
factors are selected from the group comprising VEGF and HMG proteins, and
also comprise the nucleic acids coding therefor. In an embodiment the DNA
synthesis enzymes are selected from the group comprising telomerase, and
also comprise the nucleic acids coding therefor. In an embodiment the DNA
repair enzymes are selected from the group comprising Ku-80, and also
comprise the nucleic acids coding therefor. In an embodiment the growth
factors are selected from the group comprising PDGF, EGF and M-CSF, and
also comprise the nucleic acids coding therefor. In a further embodiment
the receptors are in particular those of growth factors, whereby
preferably the growth factors are selected from the group comprising
PDGF, EGF and M-CSF, and also comprise the nucleic acids coding therefor.
In an embodiment the transcription factor is selected from the group
comprising YB-1, and also comprises the nucleic acid coding therefor. In
an embodiment the metalloproteinases are in particular matrix
metalloproteinases. In a preferred embodiment the matrix
metalloproteinases are selected from the group comprising MMP-1 and
MMP-2, and also comprise the nucleic acids coding therefor. In an
embodiment the plasminogen activators of the urokinase type are selected
from the group comprising uPa-R, and also comprise the nucleic acids
coding therefor.

[0148]SiRNA (short interfering RNA), as may be used within the present
invention, consists of two, preferably separate RNA strands which
hybridise to each other due to base complementarity which means that they
are present essentially base paired and preferably have a length of up to
50 nucleotides, preferably between 18 and 30 nucleotides, more preferably
less than 25 nucleotides and most preferably 21, 22 or 23 nucleotides,
whereby these figures refer to the single strand of the siRNA,
particularly to the length of the stretch of the single strand which
hybridises to or is base paired with a, more precisely the second single
strand. siRNA specifically induces or mediates the degradation of mRNA.
The specificity required theretofore is mediated by the sequence of the
siRNA and thus its binding site. The target sequence to be degraded is
essentially complementary to the first or to the second of the siRNA
forming strands. Although the precise mode of action is not yet clear, it
is assumed that siRNA is a biological strategy for cells in order to
inhibit distinct alleles during development and to protect themselves
against viruses. siRNA mediated RNA interference is used as a method for
the specific suppression or complete elimination of the expression of a
protein by introducing a gene specific double-stranded RNA. For higher
organisms a siRNA comprising 19 to 23 nucleotides is insofar particularly
suitable as it does not result in the activation of a non-specific
defense reaction such as an interleukin response. The direct transfection
of double-stranded RNA of 21 nucleotides having symmetrical 2-nt 3'
overhangs was suitable to mediate RNA interference in mammalian cells and
is highly efficient compared to other technologies such as ribozymes and
antisense molecules (Elbashir, S. Harborth J. Lendeckel W. Yalvcin, A.
Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells. Nature 2001, 411: 494-498). As
little as a few siRNA molecules are sufficient so as to suppress
expression of the target gene. In order to avoid the limitations of
exogenously added siRNA which particularly reside in the transient nature
of the interference phenomenon and specific delivery (delivery) of the
siRNA molecules, vectors are used in the prior art which allow for an
endogenous siRNA expression. For such purpose, for example,
oligonucleotides having a length of 64 nucleotides are introduced into
the vector which comprise the 19 nucleotide long target sequence both in
the sense and in the antisense orientation, separated by, for example, a
9 nucleotide spacer sequence. The resulting transcript folds into a
hairpin structure with a stem structure (stem) of, for example, 19 base
pairs. The loop is rapidly degraded in the cell so that a functional
siRNA molecule is generated (Brummelkamp et al., Science, 296, 550-553,
2002).

[0149]In a further aspect the present invention relates to a medicament
which comprises at least a virus according to the present invention. In a
further aspect the present invention is related to the use of the viruses
of the invention for the manufacture of a medicament. In connection
therewith the medicament is for the treatment of tumors and cancer
diseases. The tumors and cancer diseases are preferably those described
herein. Preferably the tumor and cancer diseases are those which are and
have, respectively, a resistance. Preferably the resistance is one as
described herein, particularly preferably a multiple resistance, a
multiple resistance against cytostatics and/or radiation. In a further
aspect the medicament is for restoring of the sensitivity of cells
towards cytostatics and/or radiation, whereby the cells are preferably
tumor cells which show resistance against cytostatics and/or radiation.
The restoration of the sensitivity is a process which is referred to in
English as "restoration of drug sensitivity".

[0150]In a still further embodiment the medicament further comprises at
least one pharmaceutically active compound.

[0151]In a preferred embodiment the pharmaceutically active compound is
selected from the group comprising cytokines, metalloproteinase
inhibitors, angiogenesis inhibitors, cytostatics such as Irinotecan and
CPT-11 against colorectal carcinoma and daunorubicin against leukemia,
cell cycle inhibitors such as CYC202 which inhibits CDK2/CyclinE kinase
activity and can be used against colorectal tumors (McClue S J, Int. J.
Cancer 2002, 102, 463-468) and BAY 43-9006 which inhibits Raf-1 and is,
for example, effective against mamma carcinoma (Wilhelm S M et al.,
Cancer Res. 2004, 64, 7099-7109), proteosome inhibitors such as PS-341
which inhibits the 26S proteasome activity and is used against squamous
cell carcinoma (Fribley A et al., Mol Cell Biol 2004 November; 24(22):
9695-704), recombinant antibodies directed against, for example, the EGF
receptor (Herceptin for breast carcinoma and prostate tumor; H. G. van
der Poel, European Urology 2004, 1-17; Erbitux against head and neck
tumors; Bauman M et al., Radiother. Oncol., 2004, 72, 257-266), and
inhibitors of the signal transduction cascade such as STI 571 which
represses, among others, c-kit and can be used against gastrointestinal
tumors (H. G. van der Poel, European Urology 2004, 45, 1-17), ABT-627
which is an endothelin inhibitor and which may be used, among others,
against prostate tumors (H. G. van der Poel, European Urology 2004, 45,
1-17), SU5416 which inhibits phosphorylation of the VEGF tyrosine kinase
receptor and which may be used, among others, against glioblastoma and
prostate cancer (Bischof M et al Int. J. Radiat. Oncol. Biol. Phys. 2004;
60 (4): 1220-32), ZD1839 which inhibits EGFR tyrosine activity and may be
used, among others, against prostate tumors (H. G. van der Poel, European
Urology 2004, 45, 1-17); rapamycine derivatives such as CCI-779 and
RAD001 which inhibit mTOR and can be used against prostate tumors. It is
within the present invention that the various adenoviruses described
herein and the adenoviruses to be used in accordance with the present
invention, respectively, can, in principle, be used with each and any of
the aforementioned compounds for each and any of the indication described
in connection therewith. In a particularly preferred embodiment the
indication is the one which is described for any of the previously
mentioned pharmaceutically active compounds.

[0152]The present inventor has further surprisingly found that the
efficacy of the viruses described herein and in particular the viruses
used in accordance with the present invention can be increased by using
it in combination with at least two compounds whereby each of the at
least two compounds is individually and independently selected from the
group comprising cytostatics. The compounds are in a preferred embodiment
pharmaceutically active compounds.

[0153]As used herein in a preferred embodiment, cytostatics are in
particular chemical or biological compounds which, during or after the
administration to a cell or an organism containing a or such cell, cause
that the cell no longer grows and/or no longer divides or cell division
and/or cell growth is slowed down. Cytostatics also comprise compounds
which turn into a cytostatic in the aforedescribed sense only in the cell
or in an organism containing such cell. Insofar, the term cytostatics
also comprises pre-cytostatics.

[0154]Cytostatics are grouped according to their mode of action. The
following groups are distinguished which, in principle, can all be used
within the present invention: [0155]Alkylating agents, i.e. chemical
compounds which cause their cytotoxic effect by alkylating phosphate,
amino, sulphydryl, carboxy and hydroxy groups of the nucleic acid as well
as proteins. Such compounds are often cancerogenic themselves. Typical
examples of this group of cytostatics are cis-platin and platin
derivatives, cyclophosphamide, dacarbazine, mitomycin, procarbazine.
[0156]Antimetabolites, i.e. compounds which, due to their structural
similarity or ability for binding block a metabolic process or affect the
same. Within the group of antimetabolites it is distinguished between
structurally similar antimetabolites, structure changing antimetabolites
and the indirectly acting antimetabolites. The structurally similar
antimetabolites compete due to chemical similarity with the metabolite
without exerting the function thereof. Structure changing antimetabolites
bind to the metabolites which impedes its function or resorption or
chemically modifies the metabolite. Indirectly acting antimetabolites
interfere with the function of the metabolite, for example by the binding
of ions. Typical examples of this group are folic acid antagonists such
as methotrexate, pyrimidine analogues such as fluorouracil, purine
analogues such as azathioprine and mercaptopurine. [0157]Mitosis
inhibitors, i.e. compounds which inhibit cell division. Within the group
of mitosis inhibitors it is distinguished between cell division toxins,
spindle toxins and chromosome toxins. Typical examples of this group are
taxanes and vinca alkaloids. The taxanes in turn can be divided into the
two major groups of taxoles and taxoters, whereby a particularly
preferred taxole is paclitaxel, and a particularly preferred taxoter is
docetaxel. [0158]Antibiotics having an inhibitory effect on the
DNA-dependent RNA polymerase. Typical examples are the anthracyclines,
such as, e.g., bleomycin, daunorubicin, doxorubicin and mitomycin.
[0159]Topoisomerase inhibitors, in particular topoisomerase I inhibitors.
Topoisomerase inhibitors are chemical compounds which determine the
tertiary structure of the DNA by catalysing the change of the DNA twist
number in a three stage process. Essentially, two forms of topoisomerases
are distinguished. Topoisomerases of type I cleave only a DNA strand and
are ATP-independent, whereas topoisomerase of type II cleave both strands
of a DNA, whereby they are ATP-dependent. Typical examples for
topoisomerase I inhibitors are irinotecan and topotecan, and for
topoisomersae II inhibitors etoposid and daunorubicin.

[0160]Within the present invention at least one and preferably two agents
are selected from the aforementioned group. It is, however, also within
the invention that in particular also three, four or five different
agents are selected. The following comments are made for the embodiment
of the present invention where only one and preferably two agents are
used together with the virus. These considerations are basically also
applicable to the embodiments where more than two agents are used.

[0161]Preferably the agents differ from each other such that they address
different target molecules or are described in literature as targeting
different molecules. It is within the present invention that the agent
also comprises two or more different compounds which bind to the same
target molecule. It is also within the present invention that one agent
binds to a first site of the target molecule, whereas the second agent
binds to a second site of the target molecule.

[0162]It is also within the present invention that at least two of the
agents are active using different modes of action. Active means in a
preferred embodiment that the cell growth and/or cell division inhibiting
or retarding effect of the chemical compound is mediated through a
different mode of action. In a particularly preferred embodiment the term
active means that the replication efficiency of a virus, in particular
the virus according to the present invention, of the viruses described
herein and of the viruses to be used in accordance with the present
invention, is increased compared to a scenario where one and/or both of
the agents are not used. As a measure for the efficiency of viral
replication preferably the number of viruses required for cell lysis is
used, more preferably expressed as pfu/cell.

[0163]In a particularly preferred embodiment at least one of the at least
two agents is one which increases the infectability of the cell in which
the replication of the virus is to occur, preferably is to occur in a
selective manner, preferably with the virus described herein and/or the
virus to be used in accordance with the present invention. This can,
e.g., be performed by increasing the uptake of the virus by the cell. The
uptake of the virus, in particular of adenovirus, is, for example,
mediated by the coxsackievirus-adenovirus receptor (CAR) (Mizuguchi und
Hayakawa, GENE 285, 69-77, 2002). An increased expression of CAR is, for
example, caused by trichostatin A (Vigushin et al., Clinical Cancer
Research, 7, 971-976, 2001).

[0164]In a further embodiment one of the at least two agents is one which
increases the availability of a component within the cell, whereby the
component is one which increases the replication of the virus, preferably
the virus described herein and/or the virus to be used in accordance with
the present invention.

[0165]In a further embodiment one of the at least two agents is one which
mediates the transport of YB-1 into the nucleus. Such an agent can be
selected from the group comprising topoisomerase inhibitors, alkylating
agents, antimetabolites and mitosis inhibitors. Preferred topoisomerase
inhibitors are camptothecin, irinotecan, etoposide and their respective
analogues. Preferred mitosis inhibitors are daunorubicin, doxorubicin,
paclitaxel and docetaxel. Preferred alkylating agents are cis-platin and
their analogues. Preferred antimetabolites are fluorouracil and
methotrexat.

[0166]In a particularly preferred embodiment one of the at least two
agents is one which increases the infectability of the cell, in
particular the expression of CAR, and the second of the at least two
agents is one which increases the transport of YB-1 into the nucleus,
whereby preferably as chemical compound a compound is used which exhibits
the respective required characteristic as preferably described above. An
example for the class of compounds increasing the expression of CAR are
histone deacetylase inhibitors and an example for a class of compounds
increasing the transport of YB-1 into the nucleus are topoisomerase
inhibitors.

[0167]In a further embodiment one of the at least two agents is a histone
deacylase inhibitor and the other one of the at least two agents is a
topoisomerase inhibitor.

[0168]In a further embodiment one of the at least two agents is a histone
deacylase inhibitor. A preferred histone deacylase inhibitor is one which
is selected from the group comprising trichostatin A, FR901228,
MS-27-275, NVP-LAQ824 and PXD101. Trichostatin A is, for example,
described in Vigushin et al., Clinical Cancer Research, 7, 971-976, 2001;
FR901228 is, for example, described in Kitazono et al., Cancer Res., 61,
6328-6330, 2001; MS-27-275 is described in Jaboin et al., Cancer Res.,
62, 6108-6115, 2002; PXD101 is described in Plumb et al., Mol. Cancer
Ther., 8, 721-728, 2003; NVP-LAQ824 is described in Atadja et al., Cancer
Res., 64, 689-695, 2004.

[0170]In a still further embodiment one of the at least two agents is a
topoisomerase inhibitor, preferably a topoisomerase I inhibitor. A
preferred topoisomerse inhibitor is one which is selected from the group
comprising camptothecin, irinotecan, topotecan, SN-38,
9-aminocamptothecin, 9-nitrocamptothecin, DX-895If and daunorubicin.
Irinotecan and SN-38 are, for example, described in Gilbert et al.,
Clinical Cancer Res., 9, 2940-2949, 2003; DX-895IF is described in van
Hattum et al., British Journal of Cancer, 87, 665-672, 2002; camptothecin
is described in Avemann et al., Mol. Cell. Biol., 8, 3026-3034, 1988;
9-aminocamptothecin, 9-nitrocamptothecin are described in Rajendra et
al., Cancer Res., 63, 3228-3233, 2003; daunorubicin is described in M.
Binaschi et al., Mol. Pharmacol., 51, 1053-1059.

[0171]In a preferred embodiment the topoisomerase inhibitor is selected
from the group comprising camptothecin, irinotecan, topotecan, DX-895If,
SN-38, 9-aminocamptothecin, 9-nitrocamptothecin, etoposid and
daunorubicin. These may be used against various tumors, for example,
colorectal tumors, pancreas tumors, ovary carcinomas and prostate
carcinomas. The fields of application are, among others, described by
Recchia F et al., British J. Cancer 2004, 91, 1442-1446; Cantore M et
al., Oncology 2004, 67, 93-97; Maurel J. et al., Gynecol. Oncol 2004, 95,
114-119; Amin A. et al., Urol. Oncol. 2004, 22, 398-403; Kindler H L et
al., Invest. New Drugs 2004, 22, 323-327, Ahmad T. et al., Expert Opin.
Pharmacother. 2004, 5, 2333-2340; Azzariti A. et al., Biochem Pharmacol.
2004, 68, 135-144; Le Q T et al., Clinical Cancer Res. 2004, 10,
5418-5424. It is within the present invention that the various
adenoviruses described herein and the adenoviruses to be used in
accordance with the present invention, respectively, may in principle be
used with the aforementioned compounds for each and any of the
indications described herein in connection therewith. In a particularly
preferred embodiment the indication is such as described for each of the
aforementioned pharmaceutically active compounds.

[0172]In a preferred embodiment of each and any aspect of the present
invention the further pharmaceutically active compound is selected from
the group comprising cytokines, metalloproteinase inhibitors,
angiogenesis inhibitors, cytostatics such as irinotecan and CPT-11
against colorectal carcinoma and daunorubicin against leukemia, cell
cycle inhibitors such as CYC202 which inhibits CDK2/CyclinE kinase
activity and can be used against colorectal tumors (McClue S J, Int. J.
Cancer 2002, 102, 463-468) and BAY 43-9006 which inhibits Raf-1 and is
effective against mamma carcinoma (Wilhelm S M et al., Cancer Res. 2004,
64, 7099-7109), proteosome inhibitors such as PS-341 which inhibits the
26S proteasome activity and is used against brain tumors (Yin D. et al.,
Oncogene 2004), recombinant antibodies such as against the EGF receptor
(Herceptin for breast carcinoma and prostate tumor; H. G. van der Poel,
European Urology 2004, 1-17; Erbitux against head and neck tumors; Bauman
M et al., Radiother. Oncol., 2004, 72, 257-266), and inhibitors of the
signal transduction cascade such as STI 571 which represses, among
others, c-kit and can be used against gastrointestinal tumors (H. G. van
der Poel, European Urology 2004, 45, 1-17), ABT-627 an endothelin
inhibitor which may be used, among others, against prostate tumors (H. G.
van der Poel, European Urology 2004, 45, 1-17), SU5416 which inhibits
phosphorylation of the VEGF tyrosine kinase receptor and which may be
used against head/neck tumors (Cooney et al., Cancer Chemother. Pharmacol
2004), ZD1839 which inhibits EGFR tyrosine activity and may be used,
among others, against prostate tumors (H. G. van der Poel, European
Urology 2004, 45, 1-17); rapamycin derivatives such as CCI-779 and RAD001
which inhibit mTOR and can be used against prostate tumors (H. G. van der
Poel, European Urology 2004, 45, 1-17). It is within the present
invention that the various adenoviruses described herein and the
adenoviruses to be used in accordance with the present invention,
respectively, can, in principle, be used with each and any of the
aforementioned compounds for each and any of the indications described in
connection therewith. In a particularly preferred embodiment the
indication is the one which is described for any of the previously
mentioned pharmaceutically active compounds.

[0173]In an embodiment the medicament of the invention and/or the
medicament prepared in accordance with the invention contains the virus
separated from one or several of the at least one and preferably at least
two agents which are combined with the virus in accordance with the
present invention. The agents are preferably pharmaceutically active
compounds. It is preferred that the virus is separated from any agent
which is combined with the virus. Preferably the separation is a spatial
separation. The spatial separation can be such that the virus is present
in a different package than the agent(s). Preferably the package is a
single dose unit, i.e. the virus and/or the agent(s) is/are packed as
single dose unit. The single dose units may in turn be combined to form a
package. However, it is also within the present invention that the single
dose units of the virus are combined with one or several single dose
units of one or several of the agents or packed therewith.

[0174]The kind of package depends on the way of administration as known to
the one skilled in the art. Preferably the virus will be present in a
lyophilized form or in a suitable liquid phase. Preferably, the agents
will be present in solid form, e.g. as tablets or capsules, however, are
not limited thereto. Alternatively, also the agents can be present in
liquid form.

[0175]It is within the present invention that the virus is systemically or
locally administered. It is also within the present invention that the
agents combined with the virus are systemically or locally administered
individually and independently from each other or together. Other modes
of administration are known to the persons skilled in the art.

[0176]It is within the present invention that the virus and the agents
combined with it, are administered in a chronologically separate manner
or at the same time. In connection with a chronologically separate manner
it is preferred that the agent is administered prior to the
administration of the virus. How long the agent is administered prior to
the virus depends on the kind of the agent used and is obvious for the
person skilled in the art from the mode of action of the agent used. In
case of administration of the virus in combination with at least two
agents, the administration of the at least two agents can occur at the
same or at different points in time. In connection with a chronologically
different administration the points of time again result from the modes
of action underlying the agents and can, based thereon, be determined by
the persons skilled in the art.

[0177]The above considerations, given in connection with the medicaments
according to the present invention which are also referred to herein as
pharmaceutical compositions, are roughly also applicable to any
composition, including compositions as used for the replication of
viruses, preferably for the in vitro replication of viruses in accordance
with the present invention. The above considerations are also applicable
to the kit according to the present invention and the kit to be used in
accordance with the present invention, respectively, which may apart from
the viruses described herein and the viruses to be used in accordance
with the invention, also comprise an agent or a combination of agents as
described herein. Such kits comprise the virus and/or the one or the
several agents in a form ready for use and preferably instructions for
use. Furthermore, the above embodiments apply also to the nucleic acids
as disclosed herein, and the nucleic acids used in accordance with the
present invention, and the replication systems in accordance with the
present invention and the nucleic acids coding therefor, and the
replication systems used in accordance with the present invention and the
nucleic acids coding therefor used in accordance with the present
invention, and vice versa.

[0178]The medicament in connection with which or for the manufacture of
which the adenoviruses disclosed herein are used in accordance with the
present invention, is intended to be applied, usually, in a systemic
manner, although it is also within the present invention to apply or
deliver it locally. The application is intended to infect particularly
those cells with adenoviruses and it is intended that adenoviral
replication particularly occurs therein, which are involved, preferably
in a causal manner, in the formation of a condition, typically a disease,
for the diagnosis and/or prevention and/or treatment of which the
medicament according to the present invention is used.

[0179]Such a medicament is preferably for the treatment of tumor diseases.
Those tumor diseases are particularly preferred where either YB-1 is, due
to the mechanism underlying the tumor disease, in particular due to the
underlying pathological mechanism, already located in the nucleus, which
have deregulated YB-1, or where the presence of YB-1 in the nucleus is
caused by exogenous measures whereby such exogenous measures are suitable
to transfer YB-1 into the cellular nucleus or to induce or to express it
there. The term tumor or tumor disease shall comprise herein both
malignant as well as benign tumors, and respective diseases. In an
embodiment the medicament comprises at least one further pharmaceutically
active compound. The nature and the amount of such further
pharmaceutically active compound will depend on the kind of indication
for which the medicament is used. In case the medicament is used for the
treatment and/or prevention of tumor diseases, typically cytostatics such
as, but not limited to, cis-platin and taxole, daunoblastin,
daunorubicin, adriamycin (doxorubicin) and/or mitoxantrone or others of
the cytostatics or groups of cytostatics described herein are used.

[0180]The medicament according to the invention can be present in various
formulations, preferably in a liquid form. Furthermore, the medicament
will contain adjuvants such as stabilisers, buffers, preservatives and
the like which are known to the one skilled in the art of formulations.

[0181]The present inventor has surprisingly found that the viruses of the
invention may be used with a high success rate in connection with those
tumors where YB-1 is present in the nucleus independent of the cell cycle
and those tumors which contain deregulated YB-1. Normally, YB-1 is
present in the cytoplasm, in particular also in the perinuclear plasma.
In the S phase of the cell cycle, YB-1 is in the nucleus of both normal
as well as tumor cells. This, however, is not sufficient in order to
provide a viral oncolysis using such modified adenoviruses. The
comparatively little efficacy of such attenuated viruses as described in
the prior art is ultimately based on their improper use. In other words,
such adenoviral systems, could be used, particularly also with a higher
efficacy, where the molecular biological prerequisites are given for
viral oncolysis using these attenuated or modified viruses which are
described herein. Such prerequisites are given in connection with the
aforedescribed tumor diseases, i.e. those tumor diseases the cells of
which show a nuclear localisation of YB-1 independent of the cell cycle,
or have deregulated YB-1. This kind of nuclear localisation may be caused
by the nature of the tumor itself, or by the agents according to the
present invention as described herein, including the viruses described
herein, or by other measures. The present invention thus defines a new
group of tumors and tumor diseases and thus also of patients which may
still effectively be treated using the viruses according to the present
invention and in particular also using the attenuated or modified
adenoviruses as already described in the prior art.

[0182]Without wishing to be bound in the following, it seems that
"deregulated" YB-1 is an overexpressed or phosphorylated YB-1. This is
based on the following facts. Akt which is a serine/threonine kinase,
promotes the growth of tumor cells by phosphorylation of transcription
factors and cell cycle proteins (Nicholson K M and Anderson N G, Cell.
Signal., 14, 381-395, 2002). Additionally, one has found that activated
Akt (phosphorylated Akt) correlates in a positive manner with YB-1 and
that Akt is binding to YB-1 and phosphorylates the same at position Ser
102 of the cold-shock domain (Sutherland B W et al., Oncogene, 24,
4282-4292, 2005). This is indicating that there are signal transduction
pathways which change the subcellular localisation of YB-1 and as such
immediately its function. Additionally, this phosphorylation increases
the production of proteins such as MDR and MRP, which are involved in
stress response, cell proliferation and oncogenic transformation
(Evdokimova V et al., Molecular and Cellular Biology, 26, 277-292, 2006).
The phosphorylation of YB-1 by Akt, however, weakens its Cap binding
capacity, whereby a translational activation of silent mRNA species is
made easier (Evdokimova V et al., Molecular and Cellular Biology, 26,
277-292, 2006). As Akt is not active in normal cells, YB-1 is not present
in a phosphorylated form, whereas YB-1 is deregulated, i.e.
phosphorylated and/or overexpressed in such cells.

[0183]A further group of tumors and tumor diseases and thus of patients
which may be treated using the viruses according to the invention and
thus the medicament containing the same, are those whereupon applying or
realising certain conditions it is ensured that YB-1 migrates into the
nucleus or is induced or transported thereto, including by using the
virus according to the invention or the viruses used in accordance with
the present invention. This use of the viruses in connection with these
tumors and patient groups, respectively, is based on the finding that the
induction of the viral replication is based on nuclear localisation of
YB-1 with subsequent binding of YB-1 to the E2-late promoter. This is
also true for those cells which are YB-1 nucleus-positive and/or cells
where YB-1 is present in a deregulated manner in the sense of the present
invention. Insofar the adenoviruses according to the present invention
can be used in accordance with the present invention for the treatment of
diseases and groups of patients, respectively, which have cells having
these characteristics, particularly if these cells are involved in the
development of the respective disease to be treated. A further group of
patients which can be treated by using such viruses, in particular
adenoviruses, are thus those which are YB-1 nucleus-positive as a result
of the subsequently described treatments and/or patients which have
undergone one of the measures described herein, preferably in the sense
of a treatment, or have experienced the administration of the viruses
according to the present invention or experience them together with the
administration of the virus according to the present invention. It is
within the present invention that YB-1 nucleus-positive patients are
patients which have YB-1 in the nucleus independent of the cell cycle, in
particular in a number of tumour forming cells. These measures comprise
the administration of such cytostatics as they are generally described
herein and/or as they are used in connection with tumour therapy.
Furthermore this group of measures comprises irradiation, in particular
irradiation as used in connection with tumour therapy. Irradiation means
in particular irradiation with energy-rich radiation, preferably
radioactive radiation, preferably as used in connection with tumour
therapy. A further measure is hyperthermia and the application of
hyperthermia, preferably hyperthermia as used in connection with tumour
therapy. In a particularly preferred embodiment hyperthermia is applied
locally. Finally, a further measure is hormone treatment, in particular
hormone treatment as used in connection with tumour treatment. In
connection with such hormone treatment anti-estrogens and/or
anti-androgens are used. In connection therewith, anti-estrogens such as
Tamoxifen, are particularly used in the therapy of breast cancer, and
anti-androgens as, for example, Flutamide and cyproterone acetate, are
used in the therapy of prostate cancer.

[0184]It is within the present invention that some of the tumor forming
cells which either inherently contain YB-1 in the nucleus or do so or
after induction and active introduction into the nucleus or which
comprise deregulated YB-1 in the meaning of the present disclosure.
Preferably about 5% or any percentage higher than that, i.e. 6%, 7%, 8%
etc., of the tumor forming cells are such YB-1 nucleus-positive cells or
cells in which deregulated YB-1 is present. Nuclear localisation of YB-1
may be induced by outside stress and locally applied stress,
respectively. This induction may occur through irradiation, particularly
UV-irradiation, application of cytostatics as, among others, also
disclosed herein, and hyperthermia. In connection with hyperthermia it is
important that it may be realized in a very specific manner nowadays,
particularly in a specific local manner, and that thus also a specific
nuclear transport of YB-1 into the nucleus may be caused and, because of
this, the prerequisites for replication of the adenovirus and thus of
cell and tumor lysis are given, which preferably is locally limited
(Stein U, Jurchott K, Walther W, Bergmann, S, Schlag P M, Royer H D. J
Biol Chem. 2001, 276(30):28562-9; Hu Z, Jin S, Scotto K W. J Biol Chem.
2000 Jan. 28; 275(4):2979-85; Ohga T, Uchiumi T, Makino Y, Koike K, Wada
M, Kuwano M, Kohno K. J Biol Chem. 1998, 273(11):5997-6000).

[0185]With regard to the characteristics of the cells for the lysis of
which the adenoviruses described herein are used in accordance with the
present invention, it is envisaged that these show, in an embodiment, a
resistance, preferably a multiple resistance or poly-resistance.
Resistance as used herein preferably refers to a resistance to
cytostatics and in particular to the cytostatics described herein, and/or
radiation. This multiple resistance preferably goes along with the
expression, preferably an overexpression of the membrane-bound transport
protein P-glycoprotein which is a marker for the determination of
respective cells and thus also of respective tumors and the corresponding
patient groups, respectively. The term resistance as used herein
comprises both the resistance which is also referred to as classical
resistance and which is mediated by the P-glycoprotein, as well as the
resistance which is also referred to as atypical resistance and which is
mediated by MRP or other, non-P-glycoprotein mediated resistances.
Further resistances to which it is referred to herein and which are
characteristic for the tumors and patients, respectively, to be treated
in accordance with the present invention, are those which are mediated by
the following genes, however, are not limited thereto: MDR, MRP,
topoisomerase, BCL2, glutathione-S-transferase (GST), protein kinase C
(PKC). As the effect of cytostatics is, among others, based on the
induction of apoptosis, the expression of apoptosis relevant genes plays
an important role in the formation of resistance so that also the
following factors are relevant insofar, namely Fas, the BCL2-family,
HSP70 and EGFR [Kim et al., Cancer Chemther. Pharmacol. 2002, 50,
343-352]. A further marker which correlates with the expression of YB-1
is Topoisomerase II α. Insofar, rather than or in addition to
determining YB-1 in the nucleus the expression of Topoisomerase II
α or of any of the other markers described herein, can be used in a
screening method to determine whether a patient may be treated with the
adenoviruses according to the present invention with an expectation of
success. A marker which can in principle be used similarly to the
P-glycoprotein, is MRP. A further marker at least to the extent that the
colorectal carcinoma cells or patients having a colorectal carcinoma are
afflicted or may be/are identified, is PCN (proliferating cell nuclear
antigen) (Hasan S. et al., Nature, 15, 387-391, 2001) as, for example,
described in Shibao (Shibao K et al., Int. Cancer, 83, 732-737, 1999).
Finally, at least for breast cancer and osteosarcoma cells the expression
of MDR (multiple drug resistance) is a marker in the afore-described
sense (Oda Y et al., Clin. Cancer Res., 4, 2273-2277). A further possible
marker which can be used in accordance with the present invention, is p73
(Kamiya, M., Nakazatp, Y., J Neurooncology 59, 143-149 (2002); Stiewe et
al., J. Biol. Chem., 278, 14230-14236, 2003).

[0186]It is a particular advantage of the present invention that also
those patients may be subject to treatment using in accordance with the
invention the adenoviruses described herein, which otherwise cannot be
treated anymore in the medicinal-clinical sense and where thus a further
treatment of the tumor diseases using the methods of the prior art is no
longer possible with an expectation of success, in particular where the
use of cytostatics and irradiation is no longer reasonably possible and
cannot be successfully carried out any longer in the sense of influencing
or reducing the tumor. Herein the term tumor refers in general also to
any tumor or cancer disease which either inherently contains YB-1 in the
nucleus of a cell, preferably independent of the cell cycle, or does so
upon applying exogenous measures, as disclosed herein, and/or which
contains deregulated YB-1.

[0187]Furthermore, the viruses described herein can be used, in principle,
for the treatment of tumours.

[0188]The tumours which can in particular be treated by the viruses
described herein are preferably those tumours which are selected from the
group comprising tumours of the nervous system, ocular tumours, tumours
of the skin, tumours of the soft tissue, gastrointestinal tumours,
tumours of the respiratory system, tumour of the skeleton, tumours of the
endocrine system, tumours of the female genital system, tumours of a
mammary gland, tumours of the male genital system, tumours of the urinary
outflow system, tumours of the haematopoietic system including mixed and
embryonic tumours. It is within the present invention that these tumours
are in particular resistant tumours as in particular defined herein.

[0189]The group of tumors of the nervous system preferably comprises:
[0190]1. Tumors of the skull as well as of the brain (intracranial),
preferably astrocytoma, oligodendroglioma, meningioma, neuroblastoma,
ganglioneuroma, ependymoma, schwannoglioma, neurofibroma,
haemangioblastoma, lipoma, craniopharyngioma, teratoma and chondroma;
[0191]2. Tumors of the spinal cord and of the vertebral canal, preferably
glioblastoma, meningioma, neuroblastoma, neurofibroma, osteosarcoma,
chondrosarcoma, haemangiosarcoma, fibrosarcoma and multiple myeloma; and
[0192]3. Tumors of the peripheral nerves, preferably schwannoglioma,
neurofibroma, neurofibrosarcoma and perineural fibroblastoma.

[0212]The group of the tumors of the endocrine system preferably
comprises: [0213]1. Tumors of the thyroid gland/parathyroid, preferably
adenoma and adenocarcinoma; [0214]2. Tumors of the suprarenal gland,
preferably adenoma, adenocarcinoma and pheochromocytoma
(medullosuprarenoma); [0215]3. Tumors of the hypothalamus/hypophysis,
preferably adenoma and adenocarcinoma; [0216]4. Tumors of the endocrine
pancreas, preferably insulinoma (beta cell tumor, APUDom) and
Zollinger-Ellison syndrome (gastrin secernent tumor of the delta cells of
the pancreas); and [0217]5. multiple endocrine neoplasias (MEN) and
chemodectoma.

[0236]The group of the mixed and embryonal tumors preferably comprises:
[0237]Haemangiosarcoma, thymoma and mesothelioma.

[0238]In a particularly preferred embodiment these tumors are selected
from the group comprising breast cancer, ovary carcinoma, prostate
carcinoma, osteosarcoma, glioblastoma, melanoma, small-cell lung
carcinoma and colorectal carcinoma. Further tumors are those which are
resistant as described herein, preferably those which are multiple
resistant, particularly also those tumors of the group described above.
Especially preferred tumors are also those selected from the group
comprising breast tumors, bone tumors, stomach tumors, intestinal tumors,
gallbladder tumors, pancreatic tumors, liver tumors, kidney tumors, brain
tumors, ovary tumors, tumors of the skin and of cutaneous appendages,
head/neck tumors, uterus tumors, synovial tumors, larynx tumors,
oesophageal tumors, tongue tumors and prostate tumors. It is preferred
that these tumors are, with regard to their manifestations, those which
are disclosed herein.

[0239]Further tumours which can be treated using the viruses according to
the present invention are leukaemia and metastatizing tumours, in
particular metastatizing tumours of the previously recited tumours.
Further tumours which may be treated in accordance with the present
invention, are selected from the group comprising primary tumours,
secondary tumours, tertiary tumours and metastatizing tumours. It is
preferred if the tumours comprise at least one of the following features,
namely that they have YB-1 in the nucleus independent of the cell cycle,
regardless what the reason therefore is, and/or that they comprise
deregulated YB-1. A further group of tumours which may be treated using
the viruses according to the present invention, are all of the
afore-mentioned tumours and tumours, respectively, which are described as
being treatable by using the viruses according to the present invention,
provided that they have one or several of the resistances disclosed
herein.

[0240]It is further within the present invention that also such tumours
can be treated using the viruses according to the present invention,
which do neither contain YB-1 in the nucleus, preferably independent of
the cell cycle, nor deregulated YB-1. This is realized in particular if
the viruses themselves code for YB-1. For reasons of specific expression
of YB-1 and thus of specific replication of the viruses, the expression
of the viruses is put in a preferred embodiment under the control of a
preferably highly regulated promoter. Such a promoter could be any
promoter which can be activated in a specific manner so that the viruses
can only replicate in the intended cells. Particularly preferred
promoters are in particular tumour-specific promoters and tissue-specific
promoters which are known to the ones skilled in the art. Furthermore, it
is also possible to clone the sequence coding for YB-1 into the viral
genome such that it is expressed by the adenoviral major late promoter
(MLP). This promoter is mostly active after the start of the adenoviral
replication (Tollefson A. E. et al., Journal of Virology 66, 3633-3642,
1992; Bauzon M. et al., Molecular Therapy 7, 526-534, 2003).

[0241]YB-1 belongs to the group of highly conserved factors which bind to
an inverted CAAT sequence, the so-called Y-box. They may be active in a
regulatory manner both at the level of transcription as well as
translation (Wolffe, A. P. Trends in Cell Biology 8, 318-323, 1998).

[0242]The nucleic acid coding for YB-1 which, in an embodiment of the
adenoviruses to be used in accordance with the present invention, is part
of the adenoviruses, may also comprise a nucleic acid sequence mediating
the transport of YB-1 into the nucleus. The nucleic acids, adenoviruses
and adenoviral systems according to the invention as well as the
adenoviruses known in the prior art such as, for example, Onyx-015,
AdΔ24, dl922-947, E1Ad/01/07, CB016, dl 520 and the adenoviruses
described in patent EP 0 931 830, can be used as such or in combination
with these nucleic acids in accordance with the invention in connection
therewith as adenoviruses and adenoviral systems and thus as the
corresponding nucleic acids. Suitable nucleic acid sequences which
mediate nucleus transport, are known to the ones skilled in the art and,
for example, described in (Whittaker, G. R. et al., Virology, 246, 1-23,
1998; Friedberg, E. C., TIBS 17, 347, 1992; Jans, D. A. et al., Bioessays
2000 June; 22(6): 532-44; Yoneda, Y., J. Biocehm. (Tokyo) 1997 May;
121(5): 811-7; Boulikas, T., Crit. Rev. Eukaryot. Gene Expr. 1993; 3(3):
193-227; Lyons R H, Mol. Cell Biol., 7, 2451-2456, 1987). In connection
with the nucleus transport mediating nucleic acid sequences, different
principles can be used. One such principle may, for example, be that YB-1
is formed as a fusion protein together with a signal peptide and is
introduced into the nucleus and that the replication of the adenoviruses
according to the present invention thus occurs.

[0243]A further principle which may be realised in the design of the
adenoviruses used in accordance with the invention, is that YB-1 can be
provided with a transporter sequence which, preferably starting from
synthesis in the cytoplasma, introduces YB-1 into the cell nucleus or
which translocates YB-1 into the cell nucleus, and promotes viral
replication there. An example for a particularly effective nucleic acid
sequence mediating nucleus transport is the TAT sequence of HIV which is,
among other suitable nucleic acid sequences of that type described in
Efthymiadis, A., Briggs, L J, Jans, D A., JBC 273, 1623-1628, 1998. It is
within the present invention that the adenoviruses which are used in
accordance with the present invention, comprise nucleic acid sequences
which code for peptides coding for nuclear transportation.

[0244]It is within the present invention that YB-1 is present in its full
length, particularly in a form which corresponds to the wildtype of YB-1.
It is within the present invention that YB-1 is used or present as a
derivative, such as, e.g., in a shortened or truncated form. A YB-1
derivative as used or present within the present invention, is a YB-1
which is capable of binding to the E2-late promoter and thus activates
gene expression of the adenoviral E2 region. Such derivatives
particularly comprise the YB-1 derivatives disclosed herein. Further
derivatives may be generated by deletion of single or several amino acids
at the N-terminus, at the C-terminus or within the amino acid sequence.
It is within the present invention that YB-1 fragments are also used as
YB-1 proteins in the meaning of the present invention. Various YB-1
fragments are disclosed in the paper of Jurchott K et al. [JBC 2003, 278,
27988-27996] which are characterized by deletions in the C-terminus and
the N terminus. The distribution of the various YB-1 fragments indicated
that both the cold-shock domain (CSD) as well as the C-terminus are
important for the cell cycle-regulated transport of YB-1 into the
nucleus. It is thus within the present invention that a truncated YB-1
(which is also referred to herein as YB-1 protein) is migrating in a
better way into the nucleus in combination with the expression of E1B55k
and E4orf6 in accordance with the present invention and thus induces a
stronger CPE without necessarily binding better to the E2-late promoter
compared to native YB-1, whereby it cannot be excluded that also a
truncated YB-1 is migrating better into the nucleus and exhibits both
activities, i.e. induces CPE and binds to the E2-late promoter. Finally,
such truncated YB-1 fragments can also better migrate into the nucleus
and bind better to the E2-late promoter better without inducing a better
CPE. It is also within the present invention that truncated YB-1 proteins
or fragments comprise further sequences such as described herein in
connection with the full length YB-1, in particular cellular localization
signal sequences (NLS) and the like.

[0245]The invention is related in a further aspect to a method for the
screening of patients which may be treated by using the viruses according
to the present invention and/or by using the viruses and means and
medicaments, respectively, described herein in accordance with the
present invention, whereby the method comprises the following steps:
[0246]Analysing a sample of the tumor tissue and [0247]Determining
whether YB-1 is localised in the nucleus independent of the cell cycle,
or whether the cell contains deregulated/overexpressed YB-1.

[0248]Instead of or in addition to YB-1 also the presence of the
afore-described markers which represent an alternative to YB-1, can be
assessed.

[0249]In an embodiment of the method according to the invention it is
contemplated that the analysis of the tumor tissue occurs by means of an
agent which is selected from the group comprising antibodies against
YB-1, specifically binding peptides, aptamers against YB-1, spiegelmers
against YB-1 as well as anticalines against YB-1. In principle, the same
kind of agents can also be made and used, respectively, for the
respective markers which represent an alternative to YB-1. The
manufacture of antibodies, in particular monoclonal antibodies, is known
to the ones skilled in the art. A further agent for specific detection of
YB-1 or the markers are peptides which bind with a high affinity to their
target structures, in the present case YB-1 or said markers. In the prior
art methods are known such as, for example, phage-display, in order to
generate such peptides. For such purpose, it is started from a peptide
library whereby the individual peptides have a length of about 8 to 20
amino acids and the size of the library is about 102 to 1018,
preferably 108 to 1015 different peptides. A particular form of
target molecule binding polypeptides are the so-called anticalines which
are, for example, described in German patent application DE 197 42 706.

[0250]A further agent for specifically binding to YB-1 or the
corresponding alternative markers disclosed herein and thus for the
detection of a cell cycle independent localisation of YB-1 in the
nucleus, are the so-called aptamers, i.e. D-nucleic acids, which, based
on RNA or DNA, are present as either a single strand or a double strand
and specifically bind to a target molecule. The generation of aptamers
is, for example, described in European patent EP 0 533 838. A special
embodiment of aptamers are the so-called aptazymes which, for example,
are described by Piganeau, N. et al. (2000), Angew. Chem. Int. Ed., 39,
no. 29, pages 4369-4373. They are a particular embodiment of aptamers
insofar as they comprise apart from the aptamer moiety a ribozyme moiety
and, upon binding or release of the target molecule binding to the
aptamer moiety, the ribozyme moiety becomes catalyctically active and
cleaves a nucleic acid substrate which goes along with generation of a
signal.

[0251]A further form of the aptamers are the so-called spiegelmers, i.e.
target molecule binding nucleic acids which consist of L-nucleic acids.
The method for the generation of such spiegelmers is, for example,
described in WO 98/08856.

[0252]The sample of the tumor tissue can be obtained by punctuation or
surgery. The assessment whether YB-1 is located in the nucleus
independent of the cell cycle is frequently done by the use of
microscopic techniques and/or immunohistoanalysis, typically using the
antibody or any of the further agents described above. Further methods
for the detection of YB-1 in the nucleus and that its localisation there
is independent of the cell cycle, are known to the one skilled in the
art. For example, localisation of YB-1 can easily be detected when
scanning tissue slices stained against YB-1. The frequency of YB-1 being
in the nucleus is already an indication that the localisation in the
nucleus is independent of the cell cycle. A further possibility for cell
cycle independent detection of YB-1 in the nucleus is the staining
against YB-1 and assessment whether YB-1 is localised in the nucleus and
determining the phase of the cells. This and the detection of YB-1,
respectively, however, can also be performed using the afore-mentioned
agents directed against YB-1. The detection of the agents is done by
procedures known to the persons skilled in the art. Because said agents
are specifically directed against YB-1 and insofar do not bind to other
structures within the sample to be analysed, particularly other
structures of the cells, both the localisation of said agents by means of
a suitable labelling of the agents and due to their specific binding to
YB-1, both the localisation of YB-1 can be detected and assessed
accordingly. Methods for the labelling of the agents are known to the
ones skilled in the art.

[0253]It is within the present invention that the viruses described
herein, whether they are the viruses according to the present invention,
or whether they are the viruses to be used in accordance with the present
invention, may also be used in connection with diseases, in particular
tumor diseases and more preferably tumor diseases where at least part of
the tumor cells exhibit a multiple resistance, in particular a multidrug
resistance, whereby YB-1 is present in a deregulated form. This applies
also to each and any of the other aspects as described herein in
connection with cells and tumors, provided that they refer to the cells
and diseases where YB-1 is present in the nucleus, preferably independent
of the cell cycle.

[0254]Although the viruses in accordance with the present invention and as
disclosed herein, are preferably adenoviruses the insights, methods and
uses, nucleic acids, proteins, replication systems and the like are not
limited to adenoviruses.

[0255]The aforementioned considerations, including any use as well as the
generation of the adenoviruses and adenoviral systems, apply equally to
the nucleic acids coding therefore and vice versa.

[0256]In connection with the present invention it is possible that the
adenoviruses which are used in accordance with the present invention and
the nucleic acids coding therefore, respectively, is any corresponding
viral nucleic acid which result in a replication event per se or in
combination with further nucleic acid sequences. It is possible, as
explained herein, that by means of helper viruses the sequences and/or
gene products are provided which are necessary for replication. To the
extent it is referred to coding nucleic acid sequences and to the extent
they are nucleic acid sequences which are known, it is within the present
invention that not only the identical sequence, but also sequences
derived therefrom, are used. Derived sequences are in particularly those
sequences which still result in a gene product, either nucleic acid or a
polypeptide having a function which corresponds to one or the function of
the non-derived sequence. This can be determined by simple routine tests
known to the one skilled in the art. An example for such derived nucleic
acid sequences are nucleic acid sequences which code for the same gene
product, in particularly the same amino acid sequence, however, due to
the degeneracy of the genetic code, exhibit a different base sequence.

[0257]It is within the present invention that the viruses in accordance
with the present invention are present as replication systems with or
without helper viruses.

[0258]It is further within the present invention that in case of such
adenoviral replication system according to the invention the adenoviral
nucleic acid and/or the nucleic acid of the helper virus is present as a
replicable vector.

[0259]It is further within the present invention that the nucleic acid(s)
coding for the adenoviruses which are used in accordance with the present
invention, is/are present in a vector, preferably an expression vector
and that this expression vector is used in accordance with the present
invention.

[0260]In a further aspect the present invention is also related to a
vector group comprising at least two vectors, whereby the vector group in
its entirety comprises a viral replication system as described herein,
and the vector group is used in accordance with the present invention. It
is within the invention that each of the components of the viral
replication system is present on a separate vector, preferably an
expression vector.

[0261]Finally, the present invention is related in a further aspect to the
use of a cell which contains one or several of the nucleic acids which
code for the viruses described herein and which are to be used in
accordance with the present invention, and/or a corresponding adenoviral
replication system and/or a corresponding vector and/or a vector group
according to the invention, for the very same purpose as described herein
for the various viruses.

[0262]The above described constructs of viruses and in particular their
nucleic acids and the nucleic acids coding therefor, respectively, may
also be introduced in a multipartite form into a cell, preferably a
tumour cell, whereby, due to the presence of the various individual
components, these components act together as if the individual components
were derived from a single nucleic acid and a single or several viruses,
respectively.

[0263]The nucleic acids which are used in accordance with the invention
and which code for viruses, viral systems or parts thereof, may also be
present as vectors. Preferably these vectors are viral vectors. In case
the nucleic acids comprise viral nucleic acids, preferably the virus
particle is the vector. It is, however, also within the present invention
that said nucleic acids are present in a plasmid vector. In each case the
vector comprises elements which allow for and control the propagation of
inserted nucleic acid, i.e. replication and the optional expression of
the inserted nucleic acid. Suitable vectors, preferably expression
vectors, and respective elements are known to the ones skilled in the art
and, for example, described in Grunhaus, A., Horwitz, M. S., 1994,
Adenoviruses as cloning vectors. In Rice, C., editor, Seminars in
Virology, London: Saunders Scientific Publications.

[0264]The above described embodiment where the various elements of said
nucleic acids are not necessarily contained in only one vector, takes
into consideration the aspect of the invention related to the vector
group. A vector group comprises accordingly at least two vectors.
Otherwise the general statements made herein in relation to vectors are
applicable to the vectors and the vector group, respectively.

[0265]The viruses used in accordance with the present invention are
characterised by the various nucleic acids and gene products,
respectively, disclosed herein and may otherwise comprise all those
elements known to the person skilled in the art and as is the case in
particular with wildtype adenoviruses (Shenk, T.: Adenoviridae: The virus
and their replication. Fields Virology, vol. 3, editors Fields, B. N.,
Knipe, D. M., Howley, P. M. et al., Lippincott-Raven Publishers,
Philadelphia, 1996, chapter 67).

[0266]In a further aspect the present invention is related to a method for
the treatment of tumor diseases comprising the administration of a virus
according to the present invention, such nucleic acid, vectors,
replication systems, medicaments or pharmaceutical compositions. The
tumor disease is one as disclosed herein. The patient is in need of such
treatment and is preferably a patient selected from the groups of
patients disclosed herein.

[0267]It is within the present invention that, if not indicated to the
contrary, the features and embodiments disclosed for the respective
viruses, nucleic acids, vectors, replication systems, medicaments and
pharmaceutical compositions, each according to the invention, and those
of the viruses, nucleic acids, vectors, replication systems, medicaments
and pharmaceutical compositions to be used in accordance with the
invention, are also applicable to each and any of the other aspects of
the present invention and vice versa.

[0268]In the following the present invention shall be further illustrated
by reference to the figures and examples from which new features,
embodiments and advantages may be taken.

[0269]FIG. 1 is a schematic representation of the regulation of the E2
region of adenoviruses by the promoters E2 late and E2 early by means of
E2F and YB-1;

[0270]FIG. 2 is a schematic representation of the design of the adenovirus
of the wildtype;

[0271]FIG. 3 is a schematic representation of the adenovirus Xvir
05/promoter according to the invention which expression protein IX under
the control of the E2 late promoter;

[0272]FIG. 4 is a schematic representation of the adenovirus Xvir
05/E1A12S of the invention which expresses protein IX as part of the
E1B55K reading frame under the control of E1A12S;

[0273]FIG. 5 is a schematic representation of an adenovirus Xvir 05/E1B19K
according to the invention which expresses protein IX under the control
of E1B19K;

[0274]FIG. 5a is a schematic representation of the adenovirus Xvir
05/E3-IX according to the invention which expresses protein IX under the
control of the E3 promoter;

[0275]FIG. 6 is a schematic representation of the wildtype adenovirus and
the adenovirus Xvir 05 according to the invention which is an embodiment
of the virus Xvir 05/E1B19K;

[0276]FIG. 7 is a schematic representation of the wildtype adenovirus and
the adenovirus Xvir 05/protein IX according to the invention which is an
embodiment of the virus Xvir 05/E1A12S;

[0277]FIG. 8 is a schematic representation of the wildtype adenovirus and
the adenovirus Xvir 05/01 according to the invention which is an
embodiment of the virus Xvir 05/protein IX;

[0278]FIG. 9 is a schematic representation of the wildtype adenovirus and
the adenovirus Xvir 05/02 according to the invention which is a further
embodiment of the virus Xvir 05/protein IX;

[0279]FIG. 10 is the result of a Northern blot analysis for the detection
of protein IX; and

[0280]FIG. 11 is a schematic representation of the design of the oncolytic
adenovirus Xvir03-3'UTR.

[0281]FIG. 1 is a schematic representation of the regulation of the E2
region of adenovirus by the promoters E2-late and E2-early by means of
E2F and YB-1. In FIG. 1 the involved promoters, namely the E2-early and
E2-late promoters, are represented with regard to the binding and
activation, respectively, by means of E2F and YB-1. The wildtype E1A
protein is interfering with the binding of E2F to retinoblastoma protein
Rb. The thus released E2F is binding to the E2 early promoter and thus
induces adenoviral replication. After 8-12 h a so-called switch occurs to
the E2-late promoter. This is only possible upon the translocation of
YB-1 from the cytoplasma into the nucleus. After nuclear translocation
YB-1 activates the E2 gene expression through the binding to the E2-late
promoter.

[0282]The binding mechanism of E2F/RB and the E1A mediated release of E2F
is substantially different from the mechanism underlying the present
invention. The release of E2F from the Rb protein is not, as assumed in
the prior art, an important, not to say the decisive step of adenoviral
replication, but the nuclear localisation of the human transcription
factor YB-1. This transcription factor is present in normal cells over
the major part of the cell cycle only in the cytoplasm. After infection
with an adenovirus it is induced in the nucleus under certain conditions
or is already present in the nucleus in distinct cellular systems such as
distinct tumor diseases, i.e. including, but not limited to, breast
cancer, ovarian carcinoma, prostate cancer, osteosarcoma, glioblastoma,
melanoma, small-cell carcinoma of the lung and colorectal cancer.

EXAMPLE 1

Design of Various Protein IX Expressing Adenoviruses

[0283]Starting from the design depicted in FIG. 2 of the viral nucleic
acid of the wildtype adenovirus the various design principles disclosed
herein for the expression of protein IX by adenoviruses which replicate
in a YB-1 dependent manner, are realised and are depicted in FIGS. 3, 4,
5 and 5a. All designs have in common that they are E1A13S-minus and
E1A12S-minus in the sense that they are not controlled by the natural and
the E1A promoter present in the wildtype, respectively.

[0284]The adenovirus Xvir 05/promoter as depicted in FIG. 3 is
additionally E1B19K-minus and protein IX-minus in the sense that protein
IX is not contained in the regulatory context as existing in the
wildtype, and protein IX is not expressed. Rather, the expression is
controlled by the E2-late promoter. Protein IX is cloned into the E3
region, however, may, in principle, also be cloned into the E4 region.
The genes for E2A, E2B, E4 and MLP are still present and can also be
expressed. The transporter consisting of E4orf6 and E1B55K is formed by
the cassette E4orf6-IRES-E1B55K which is under the control of the CMV
promoter. The respective cassette has been cloned into the E1 region,
however, could also be cloned into other regions such as, for example,
the E3 or E4 region.

[0285]The adenovirus of the adenovirus Xvir05/E1A12S as depicted in FIG. 4
is additionally E1B19K-minus and protein IX-minus in the sense that
protein IX is not present in the regulatory context existing in the
wildtype and protein IX is not expressed. Rather, the expression is
caused by E1A12S which is controlled by the E2-late promoter which
results in the activation of the reading frame of protein IX which is
contained in the E1B55K coding region. Protein E1A12S has been cloned
into the E3 region, however, may also be cloned into the E4 region. The
genes for E2A, E2B, E4 and MLP are still present and can also be
expressed. The transporter consisting of E4orf6 and E1B55K is formed by
the cassette E4orf6-IRES-E1B55K which is under the control of the CMV
promoter. The respective cassette has been cloned into the E1 region,
however, could also be cloned into different regions such as into the E3
or E4 region.

[0286]The adenovirus of adenovirus Xvir 05/E1B19K as depicted in FIG. 5 is
additionally E1B19K-minus and protein IX-minus in the sense that protein
IX is not contained in the regulatory context as existing in the
wildtype. Rather the expression is controlled by protein E1B19K which is
expressed under the control of the CMV promoter and which allows the
expression of the reading frame of protein IX which is contained in the
E1B55K reading frame. The genes for E2A, E2B, E3, E4 and MLP are still
present and can also be expressed. The transporter consisting of E4orf6
and E1B55K is formed by the cassette E4orf6-RSV-promoter-E1B region which
is under the control of the CMV promoter. The respective cassette has
been cloned into the E1 region, however, could also be cloned into
different regions such as, for example, the E3 or E4 region.

[0287]The adenovirus of adenovirus Xvir05/E3-IX as depicted in FIG. 5a is
additionally E1B19K-minus and protein IX-minus in the sense that protein
E1B19K is not contained in the regulatory context of the wildtype and
that protein IX is not expressed. Rather the expression is controlled by
the natural E3 promoter. The genes for E2A, E2B, E4 and MLP are still
present and can also be expressed. The transporter consisting of E4orf6
and E1B55K is formed by the cassette E4orf6-IRES-E1B55K which is under
the control of the CMV promoter. The respective cassette has been cloned
into the E1 region, however, could also be cloned into other regions such
as the E3 or E4 region.

[0288]FIGS. 6 to 9 represent further embodiments of the adenoviruses of
the invention.

[0289]The virus depicted in FIG. 6 is a further development of adenovirus
Xvir 05/E1B19K as depicted in FIG. 5. In addition to Xvir05/E1B19K this
virus comprises a cassette which is under the control of the E2-late
promoter and which comprises E1A12S and YB-1 and a nucleic acid,
respectively, coding each and individually therefor, whereby both reading
frames are separated from each other by an IRES. In an embodiment the
YB-1 coding nucleic acid is not contained in the cassette. The nucleic
acid for YB-1 expressed by the virus results in a more pronounced
replication in cells with deregulated YB-1.

[0290]The adenovirus depicted in FIG. 8 is a further development of the
adenovirus depicted in FIG. 6, whereby the cassette which is under the
control of the E2-late promoter comprises E1A12S and YB-1 and a nucleic
acid, respectively, coding each and individually therefor, which is
cloned into the E4 region and various transgenes are cloned into the E3
region under the control of the E3 promoter such as, for example,
apoptosis-inducing genes, prodrug genes, siRNA, tumor suppressor genes
and cytokines. Alternatively, the various transgenes disclosed herein may
be cloned into this region.

[0291]Finally, the adenovirus of the invention as depicted in FIG. 9 is a
further development of the adenovirus depicted in FIG. 8, whereby here
additionally the RGD motif which is advantageous for the targeting of the
viruses, has been incorporated by cloning. It can be found in the
adenoviral genome in the fibre protein approximately at positions
32675-32685. These variations of the specific position details is caused
by the fact that the sequences of the wildtype adenovirus are different
and have different lengths in the various data banks or data bank
entries.

[0292]The adenovirus of the invention depicted in FIG. 7 is based on the
virus depicted in FIG. 3. In contrast thereto, this adenovirus, however,
does not comprise a cassette consisting of E4orf6 and E1B55K, but both
are controlled by separate promoters, namely the CMV promoter and the RSV
promoter. The cloning had been made into the E1 region. Additionally, the
adenovirus comprises apart from the nucleic acid coding for E1A12S which
is under the control of the E2-late promoter, still a nucleic acid, which
codes for protein IX, which is separated from the one of E1A12S by an
IRES. Also this cassette could in principle be designed without the
nucleic acid coding for protein IX. In a further embodiment the cassette
is cloned into the E4 region. Finally, also this virus could comprise in
the E3 or the E4 region transgenes as described in connection with the
virus depicted in FIG. 8. In a further embodiment of this adenovirus the
RGD motif is contained.

EXAMPLE 2

Detection of Protein IX Expression

[0293]This experiment was carried out in order to confirm the relevance of
the expression of protein IX for effective particle formation in YB-1
mediated replication. For such purpose the oncolytic, YB-1 dependent
replicating adenovirus Xvir 03-03'UTR was used which is described in the
prior art and which is depicted in FIG. 11.

[0294]When carrying out the experiment it was proceeded as follows: Per 10
cm dish 106 293 and 257RDB cells were plated. At the next day the
cells were, as depicted in FIG. 11, either not infected (K), infected
with the wildtype adenovirus or with Xvir03. The infection was made in
1.5 ml serum-free DMEM medium for 1 h at 37° C. Subsequently the
infection medium was removed and replaced by 10 ml whole medium (10%
FCS/DMEM). After 24-48 h RNA was isolated. Subsequently, a Northern blot
analysis was performed. For such purpose, each 10 μg RNA were
separated by electrophoresis in an agarose gel containing 3%
formaldehyde, subsequently blotted onto a nylon membrane and hybridised
against a 386 bp probe. As probe which was generated by PCR, a P32
labelled probe was used targeting protein IX. The following primer was
used for the PCR: 5'-TATTTGACAACGCG; 5'-TTTTAAACCGCATTGGG. The position
of the probe in the adenovirus genome of the wildtype is between position
3648 and 4033. The virus used is Xvir 03 which does not show expression
of protein IX.

[0296]As may be taken from FIG. 11, virus Xvir03-03'UTR shows a reduced
expression in tumor cells 257RDB compared to wildtype adenovirus. In 293
cells which express E1A and E1B proteins, among others also the E1B19K
protein, sufficient protein IX is expressed.

[0297]In vector Xvir05 the expression of, among others, the viral proteins
E4orf6 and E1B55k is provided by the expression cassettes CMV-E4orf6 and
RSV-E1B-region. This results in translocation of YB-1 into the nucleus.
The E1A12S gene product, as well as the YB-1 gene product, under the
control of the E2-late promoter, additionally promote viral replication.
Additionally, the virus is capable of inhibiting the expression of the
ABC transporters MRP and MDR1. Additionally, the proteins E1B19K and
protein IX are expressed as part of the cassette RSV-E1B region.

[0298]The vector Xvir05-protein IX is a further vector development. There,
the expression of the adenoviral protein IX which is present in the
expression cassette E2late-E1A12S-IRES-protein IX is ensured. The vector
does not contain the whole E1B region, but only the open reading frame of
E1B55k.

[0299]The complete E1B region, i.e. E1B19k, E1B55k and protein IX are
controlled by a viral, non-adenoviral promoter in case of vector
Xvir05/01, such as the RSV promoter. The expression cassette
E2-late-E1A12S-IRES-YB-1 is present in the E4 region. Thus specific
therapeutic transgenes can be cloned into the E3 region. The E3 deletion
is such that the adenoviral ADP protein "adenoviral death protein" can
still be expressed. Additionally, the expression of E1A12S and E1B19k
results in the expression of protein IX.

[0300]The vector Xvir05/02 additionally comprises an RGD motif in the H
loop of the fibre knob in order to provide for better infection rates.

[0301]The preparation of the virus was as follows:

Modification of the Rescue Plasmid pAdEASY (Qbiogene)Use of the Shuttle
Vector pShuttle-AdEASY for the Preparation of a ΔE3E4 Shuttle
Vector

[0302]First a CMV promoter was introduced into the available vector
pShuttle-AdEASY and a bovine growth hormone polyadenylation signal cloned
into it. For such purpose the plasmid was digested with EcoRI, the ends
made blunt-ended by filling with T4 polymerase and dNTPs, the backbone
dephosphorylated and the two resulting cleavage products religated. By
this procedure the restriction recognition sequences for EcoRI were
destroyed. The plasmid thus obtained was referred to as
pShuttle(-EcoRI)-AdEASY.

[0303]Subsequently, the cassette CMV-MCS-polyA was excised from the
pShuttle of Clontech by using MfeI and EcoRI, the ends made blunt-ended
and cloned into the vector pShuttle(-EcoRI)-AdEASY which was linearised
with XbaI, made blunt-ended and dephosphorylated for such purpose. The
plasmid CMV-MCS-PolyA-pShuttle-AdEASY was thus generated.

[0304]For the manipulation of the E3 and E4 region the ΔE3E4 region
of the plasmid pAdEASY was cloned with SpeI and PacI into plasmid
CMV-MCS-PolyA-pShuttle-AdEASY and thus the plasmid ΔE3E4
pShuttle-AdEASY prepared. By restriction with NdeI and religation one of
the two NdeI cleavage sites was deleted and thus also the multiple
cloning site from the plasmid. By this procedure plasmid
ΔE3E4-pShuttle(-NdeI)-AdEASY was obtained.

E4 Manipulation

[0305]In order to provide space for potential therapeutic transgenes and
in order to avoid an undesired homologous recombination, the E4 region in
plasmid ΔE3E4-pShuttle(-NdeI)-AdEASY was specifically deleted. For
such purpose the E4orf6 region was truncated by about 634 bp by means of
cleavage with PstI and
religation=ΔE3E4ΔORF6-pShuttle(-NdeI)-AdEASY. The respective
deletions can be made in other systems for the generation of recombinant
adenoviruses by a person skilled in the art.

Cloning of the RGD Motif in ΔE3E4ΔORF6-pShuttle(-NdeI)-AdEASY

[0306]For an improved infectivity and referring to Dmitriev et al. 1998
(An Adenovirus Vector with Genetically Modified Fibers Demonstrates
Expanded Tropism via Utilization of a Coxsackievirus and Adenovirus
Receptor-Independent Cell Entry Mechanism) the HI loop of the fibre knob
domain was modified: The respective region was amplified using the
primers RGD-Hpa fw (5'-GAGgttaacCTAAGCACTGCCAAG-3'), RGD-EcoRV rev
(5'-CATAGAGTATGCAGATATCGTTAGTGTTACAGGTTTAGTTTTG-3') as well as RGD-EcoRV
fw (5'-GTAACACTAACGATATCTGCATACTCTATGTCATTTTCATGG-3') and RGD-BfrI rev
(5'-CAGCGACATGAActtaagTGAGCTGC-3') and thus an EcoRV cleavage site
generated. Into this cleavage site paired oligonucleotides were cloned
which coded for Arg-Gly-Asp (RGD) peptide: RGD-Oligo 1
(5'-CACACTAAACGGTACACAGGAAACAGGAGACACAACTTGTGACTGCCGCGGAGA
CTGTTTCTGCCC-3') and RGD-Oligo 2 (5'-GGGCAGAAACAG TCTCCGCGGCAGTCA
CAAGTTGTGTCTCCTGTTTCCTGTGTACCGTTTAGTGTG-3'). By cloning into the HpaI and
BfrI cleavage site in the ΔE3E4ΔORF6-pShuttle(-NdeI)-AdEASY
ΔE3-RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY was generated. The RGD
motif is present in the HI loop of the fibre knob domain.

Cloning of the E3a Region into the ΔE3 Region of
ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY.

[0307]For such purpose the vector pcDNA3.1(+) of Invitrogen was digested
with BglII and BamHI, whereby the CMV promoter was removed and the vector
religated (pcDNA3.1(+) without CMV=oCMV). To the SpeI and XhoI
restriction sites of the pcDNA3.1(+) oCMV vector the 2709 bp fragment
which was dissected with SpeI (27083 bp) and XhoI (29792 bp) from the
wildtype virus DNA, was cloned into (pcDNA3.1(+) oCMV/E3aXhoI).
Alternatively, one may cut at the 3' end with HpaI (30570 bp) rather than
with XhoI. For such purpose the vector pcDNA3.1 (+) oCMV is then digested
with SpeI and EcoRV and the adenoviral SpeI-HpaI-fragment cloned therein
(pcDNA3.1(+) oCMV/E3aHpaI). A further option is provided by the 2718 bp
EcoRI fragment from the adenovirus wildtype DNA (positions 27332 bp and
30050 bp) which is cloned into pcDNA3.1 (+) oCMV which has been opened
using EcoRI (pcDNA3.1(+) oCMV/E3aEcoRI).

[0308]Using pcDNA3.1(+) oCMV/E3a the E3a region could be cloned into the
vector ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY: The shuttle
vector ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY was digested
with NheI for such purpose, the ends made blunt-ended and further
digested with SpeI. The insert from pcDNA3.1(+) oCMV/E3aXhoI was cloned
into this site: The plasmid was digested with XhoI for such purpose, the
ends made blunt-ended and further digested with SpeI. The fragment thus
excised was cloned into the previously cut open plasmid
ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY.

[0309]The fragments SpeI-HpaI (position 27083 bp to 30570 bp) and EcoRI
(position 27332 bp to 30050 bp) may be prepared in a similar manner from
the respective pcDNA3.1(+) oCMV/E3a constructs and cloned.

[0310]Alternatively, the E3a region may be amplified by PCR using the
primers E3a forward (SpeI) 5'-CTTAAGGACTAGTTTCGCGC-3' and E3a reverse
(XhoI, NheI) 5'-CAAGCTAGCTCGAGGAATCATG-3' using the adenovirus type 5
wildtype DNA as template. With the E3a reverse primer a NheI cleavage
site is generated. The amplificate is restricted with SpeI and NheI and
cloned into the vector ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY
also digested with SpeI and NheI.

For the SpeI-HpaI Fragment

[0311]Alternatively, the E3a region can be amplified by PCR using the
primers E3a forward (SpeI) 5'-CTTAAGGACTAGTTTCGCGC-3' and E3a reverse
(HpaI, NheI) 5'-CACGCTAGCAAGTTAACCATGTCTTGG-3' using the adenovirus type
5 wildtype DNA as template. Using the E3a reverse primer an NheI cleavage
site is thus generated. The amplificate is restricted with SpeI and NheI
and cloned into the vector
ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY which is also opened by
SpeI and NheI.

For the EcoRI Fragment

[0312]Alternatively, the E3a region can be amplified by PCR using the
primers E3a forward (EcoRI) 5'-GAAACCGAATTCTCTTGGAAC-3' and E3a reverse
(NheI, EcoRI) 5'-GAATTCTAGCTAGCTCAGCTATAG-3' using the adenovirus type 5
wildtype DNA as template. Using the E3a reverse primer an NheI cleavage
site is thus generated. The amplificate is restricted with EcoRI and NheI
and cloned into the vector
ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY also cut open with
EcoRI and NheI.

[0313]The cloning of the E3a region from pcDNA3.1(+) oCMV/E3a in
ΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY
E3aΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY was generated.

[0314]The thus cloned region comprises the E3 region until after the open
reading frame for the E3 ADP (position 29772 bp) and thus the E3
promoter, the complete E3A region with the polyadenylation signal, the
transcription start and the open reading frame for 12.5 K, E3 6.7 K, E3
gp19 K and E3 ADP.

[0316]Further deletions between the E3 promoter and the open reading frame
for the ADP are possible with plasmid pcDNA3.1(+) oCMV/E3a: By further
restrictions between position 27596 bp and 29355 bp, for example with
EcoRII, BsiWI, DraI, MunI, the open reading frames for 6.7 K and gp19 K
which are present in between, may be removed and thus provided up to 1.8
kb of additional space for the incorporation of further transgenes. By a
respective restriction the above noted E3A amplificates can also be
truncated and be cloned as previously described.

Cloning of the Second Expression Cassette E1a 12S Under the Control of the
E2 Late Promoter.

[0317]First, the E2 late promoter was cloned as paired oligonucleotide
(upper primer 5'-TCGAGCTCCGCATTTGGCGGGCGGGATTGGTCTTCGTAGAACCTAATCTCGTGGG
CGTGGTAGTCCTCAGGTACAAAT-3' and lower primer
5'-AGCTTATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACC
AATCCCGCCCGCCAAATGCGGAGC-3' in the HindIII and BglII cleavage site of the
pGL3-enhancer plasmid of Promega (pGL3-E2Late).

[0318]Subsequently, the luciferase gene was excised with NcoI and XbaI,
the ends made blunt-ended and T ends added. At the thus opened site the
transgene E1A 12S which was amplified by RT-PCR using the primers E1a 12S
forward 5'-ATGGCCGCCAGTCTTTTG-3' and E1a 12S reverse
5'-TTATGGCCTGGGGCGTTTAC-3', was introduced by TA cloning.

[0319]By doing so, the cassette contains the E2-late promoter, the open
reading frame E1a-12S and the SV-40 late polyadenylation signal of the
vector pGL3.

[0320]This cassette was excised with PvuI and ClaI, the ends made
blunt-ended and can now alternatively be cloned into the E3a region which
was deleted by EcoRII, BsiWI, DraI, MunI (after removal of the open
reading frames for E3 6.7 K and gp19 K as above) or into the deletion of
the E4ORF6, for example into the blunt-ended and phosphorylated BfrI
cleavage site.

Cloning of the Second Expression Cassette E1a 12S with YB-1 Under the
Control of the E2Late Promoter

[0322]The amplificates E1a 12S (as above) and the IRES element
(pCITE-4a(+) of Novagen as template, IRES
forward=5'-TCCGGTTATTTTCCACCATATTGC-3' and IRES
reverse=5'-TTATCATCGTGTTTTTCAAAGG-3') were one after the other cloned
into the multiple cloning site of the pcDNA3.1(+) vector (Invitrogen).
For such purpose the E1a-12S amplificate was introduced into the
blunt-ended BamHI cleavage site by TA cloning. Subsequently, the plasmid
E1a-12S was linearised in pcDNA3.1(+) with EcoRV, T ends added and the
amplificate for the IRES element introduced by cloning. The thus obtained
construct E1a-12S-IRES-pcDNA3.1(+) was linearised with NotI and the ends
made blunt-ended; also the YB-1-EcoRI cleavage product from the plasmid
pHVad2c CMV/S40+Yb-1 s (Stephan Bergmann) was made blunt-ended and
introduced into the dephosphorylated vector E1A-12S-IRES-pcDNA3.1(+).
Alternatively, the PCR amplificate for the open reading frame of protein
IX may be introduced into the blunt-ended NotI cleavage site of the
vector E1a-12S-IRES-pcDNA3.1(+) after addition of T ends, more
specifically using the primers IX forward 5'-ATGAGCACCAACTCGTTTG-3' and
IX reverse 5'-GTTTTAAACCGCATTGGGAGG-3'.

[0323]The cassette E1A-12S-IRES-YB-1 or E1A-12S-IRES protein IX was
excised with PmeI and cloned into the above-described plasmid pGL3-E2Late
after removal of the luciferase gene with NcoI and XbaI and blunt-ending
and dephosphorylation.

[0324]This cassette E2late-E1A-12S-IRES-YB-1 was excised with PvuI and
ClaI, the ends made blunt-ended and can now alternatively be cloned into
the EcoRII, BsiWI, DraI, MunI deleted E3a region (after removal of the
open reading frames for E3 6.7 K and gp19 K, see above), or into the
deletion of the E4ORF6, for example into the blunt-ended and
dephosphorylated BfrI cleavage site.

Generation of the Rescue Plasmid
E3a/E2Late-E1a-12S/ΔE3RGD-E4ΔORF6-pAdEASY or
E3aΔE3RGD-E4ΔORF6/E2Late-E1a-12S-pAdEASY or
E3a/E2Late-E1a-12S-IRES-YB-1/ΔE3RGD-E4ΔORF6-pAdEASY or
E3aΔE3RGD-E4ΔORF6/E2Late-E1a-12S-IRES-YB-1-pAdEASY,
Respectively

[0326]The E3aΔE3RGD-E4ΔORF6 region with the second expression
cassette E2Late-E1a-12S or E2Late-E1a-12S-IRES-YB-1 in E3a or
E4ΔORF6 was excised with SpeI and PacI from the corresponding
pShuttle plasmid E3aΔE3RGD-E4ΔORF6-pShuttle(-NdeI)-AdEASY and
cloned into the corresponding opened vector pAdEASY, which created the
new rescue vector E3a/E2Late-E1a-12S/ΔE3RGD-E4ΔORF6-pAdEASY
or E3aΔE3RGD-E4ΔORF6/E2Late-E1a-12S-pAdEASY or
E3a/E2Late-E1a-12S-IRES-YB-1/ΔE3RGD-E4ΔORF6-pAdEASY or
E3aΔE3RGD-E4ΔORF6/E2Late-E1a-12S-IRES-YB-1-pAdEASY,
respectively.

[0327]E3aΔE3RGD-E4ΔORF6-pAdEASY contains the E3a region, an
RGD motif and a deleted E4ORF6, as a second expression cassette either
E2Late-E1a-12S or E2Late-E1a-12S-IRES-YB-1 are present in E3a or
E4ΔORF6. This construct is the rescue plasmid for the introducing
of further transgenes into the E1 region by a shuttle plasmid.

Preparing the Transgene Cassette for the E1 Region

Cloning of the E1B Region

[0328]For the E1B region the adenoviral genome was restricted with XbaI
(position 1340 bp) and MunI (position 3925 bp) and the 2585 bp fragment
was cloned into the pShuttle of AdEASY into the XbaI and MunI cloning
sites which thus contains the complete E1B area (pShuttle/E1B).

[0329]Alternatively, the E1B region can be amplified by PCR with the
primers E1B forward 5'-GTGTCTAGAGAATGCAATAGTAG-3' and E1B reverse
5'-GTCAAAGAATCCAATTGTGC-3' using the adenovirus type 5 wildtype DNA as
template, be restricted with XbaI and MunI and cloned into the XbaI and
MunI cleavage sites of the pShuttle of AdEASY.

[0330]Thus the pShuttle/E1B comprises the E1B promoter, the open reading
frames for E1B19K, E1B55K and protein IX and the natural Poly-A part. The
E1B promoter was removed by means of XbaI and HpaI, the ends of the
vector made blunt-ended and replaced by the CMV promoter from pcDNA3.1(+)
of Invitrogen which was cleaved with MluI and XhoI and the ends of which
were also made blunt-ended. Alternatively, rather than the CMV promoter,
an RSV promoter or a tumor-specific and viral promoters, respectively,
for example the promoters mentioned in the patent, can control the
expression of the E1B region.

Preparing the RSV Plasmid for the Preparation of the Cassette
RSV-E4ORF6-polyA.

[0331]Plasmid pRc/RSV of Invitrogen was cleaved with XhoI, SpeI and XbaI.
The thus resulting 2810 bp and 278 bp fragments were religated such that
by doing so the F1 origin and the neomycine resistance gene (oNeo) were
removed.

[0332]The thus obtained vector pRc/RSV (oNeo) comprises only one BamHI
cleavage site into which the open reading frame for E4ORF6 from plasmid
CGN from Dobbelstein was cloned. Alternatively, the amplificate of a PCR
using the primers E4ORF6-forward 5'-ATGACTACGTCCGGCGTTCC-3' and
E4ORF6-reverse 5'-CTACATGGGGGTAGAGTC-3' can be introduced into the EcoRV
cleavage site of the vector pRc/RSV (oNeo) after adding the T ends (TA
cloning). Alternatively, rather than the RSV promoter (by excision using
MliI and HindIII), a CMV promoter (obtained from the pcDNA3.1(+) by
excision with MluI and HindIII) or a tumor-specific and viral promoter,
respectively, for example the promoters described in the patent, can
control the expression of E4orf6.

[0333]The cassette RSV-E4ORF6-polyA (the bovine growth hormone
polyadenylation signal is derived from plasmid pRC/RSV) was cleaved with
MunI, the ends made blunt-ended and further excised with XhoI from the
plasmid. The expression cassette was subsequently cloned into the vector
pShuttle/E1B which had been cleaved by NotI, made blunt-ended and
subsequently cleaved with XhoI. From this vector
RSV-E4ORF6-polyA/E1B-pShuttle-AdEASY was obtained.

Introducing the Transgenic Cassette into the Rescue Vector

[0334]The vector RSV-E4ORF6-polyA/E1B-pShuttle-AdEASY for the E1-Bereich
was linearised using Bst1107I and MroI and introduced together with the
rescue plasmid (see above) into (EC) bacteria by means of
electroporation. The adenoviral plasmid
RSV-E4ORF6-polyA/E1B-E3a/E2Late-E1a-12S/ΔE3RGD-E4ΔORF6-pAdEAS-
Y was generated (or in a corresponding manner with the other above
mentioned rescue vector variants) by homologous recombination which
resulted in virus production after transfection in HEK293 cells.

[0335]It is within the present invention and obvious for a person skilled
in the art in the light of the present disclosure that other systems,
such as, for example, pAdenoX-System of Clontech/BD Biosciences or the
system of Microbix may be used for the manufacture of the adenoviruses,
preferably recombinant adenoviruses, according to the invention, in
particular those which contain the above expression cassettes
individually and/or in any combination.

[0336]The features disclosed in the preceding description, the claims as
well as the figures may be individually or in any combination essential
for the practising of the invention in its various embodiments.